Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.     

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.     

Expression and localization of components of the histone deacetylases multiprotein repressory complexes in the mouse preimplantation embryo

DNA methylation had been implicated in the assembly of multiprotein repressory complexes that affect chromatin architecture thereby rendering genes inactive. Proteins containing methyl binding domains (MBDs) are major components of these complexes. MBD3 is a component of the HDAC associated chromatin remodeling complex Mi2/NuRD. The addition of MBD2 to the Mi2/NuRD complex creates MeCP1, a complex that is known to inactivate methylated promoters. The undermethylated state of the mouse preimplantation embryo prompted us to investigate the known repressory complexes at this developmental stage. We found individual components of Mi2/NuRD: MBD3, Mi2, HDAC1 and HDAC2 to be expressed from a very early stage of embryo development and to localize in close proximity with each other and with constitutive heterochromatin by the blastula stage. Expression of MBD2, a component of MeCP1, starts in the blastula stage. Then it is also found to be in proximity with heterochromatin (based on DAPI staining) and with MBD3, Mi2 and HDAC1. In contrast, expression of MeCP2, an MBD containing component of a third repressory complex (MeCP2/Sin3A), is not seen in the preimplantation embryo. Our results suggest that both Mi2/NuRD and MeCP1 complexes are already present at the very early stages of embryo development, while a MeCP2 complex is added to the arsenal of repressory complexes post-implantation at a stage when DNA methylation takes place.     

Time-lapse and retrospective analysis of DNA methylation in mouse preimplantation embryos by live cell imaging

Genome-wide change of DNA methylation in preimplantation embryos is known to be important for the nuclear reprogramming process. A synthetic RNA encoding enhanced green fluorescence protein fused to the methyl-CpG-binding domain and nuclear localization signal of human MBD1 was microinjected into metaphase II-arrested or fertilized oocytes, and the localization of methylated DNA was monitored by live cell imaging. Both the central part of decondensing sperm nucleus and the rim region of the nucleolus in the male pronucleus were highly DNA-methylated during pronuclear formation. The methylated paternal genome undergoing active DNA demethylation in the enlarging pronucleus was dispersed, assembled, and then migrated to the nucleolar rim. The female pronucleus contained methylated DNA predominantly in the nucleoplasm. When the localization of methylated DNA in preimplantation embryos was examined, a configurational change of methylated chromatin dramatically occurred during the transition of 2-cell to 4-cell embryos. Moreover, retrospective analysis demonstrated that a noticeable number of the oocytes reconstructed by round spermatid injection (ROSI) possess small, bright dots of methylated chromatin in the nucleoplasm of male pronucleus. These ROSI oocytes showed a significantly low rate of 2-cell formation, thus suggesting that the poor embryonic development of the ROSI oocytes may result from the abnormal localization of methylated chromatin.     

Should I stay or should I go: Protection and maintenance of DNA methylation at imprinted genes

Recent findings shed light on the coordination of two fundamental, yet mechanistically opposing, processes in the early mammalian embryo. During the oocyte-to-embryo transition and early preimplantation development nuclear reprogramming occurs. This resetting of the epigenome in maternal and paternal pronuclei to a ground state is the essential step ensuring totipotency in the zygote, the first embryonic stage. Radical, global DNA demethylation, which occurs actively in the paternal and passively in the maternal genome, is a prominent feature of nuclear reprogramming; yet, this process poses a danger to a subset of methylated sequences that must be preserved for their germline to soma inheritance. Genomic imprinting and its importance were demonstrated three decades ago by a series of experiments generating non-viable mammalian uniparental embryos. Indeed, imprinted loci, gene clusters with parent-of-origin specific gene expression patterns, must retain their differential methylation status acquired during gametogenesis throughout embryogenesis and in adult tissues. It is just recently that the molecular players that protect/maintain imprinting marks during reprogramming in preimplantation embryos have been identified, in particular, an epigenetic modifier complex formed by ZFP57 and TRIM28/KAP1. The interaction of these and other molecules with the newly formed embryonic chromatin and imprinted genes is discussed and highlighted herein.     

Should I stay or should I go: Protection and maintenance of DNA methylation at imprinted genes

Recent findings shed light on the coordination of two fundamental, yet mechanistically opposing, processes in the early mammalian embryo. During the oocyte-to-embryo transition and early preimplantation development nuclear reprogramming occurs. This resetting of the epigenome in maternal and paternal pronuclei to a ground state is the essential step ensuring totipotency in the zygote, the first embryonic stage. Radical, global DNA demethylation, which occurs actively in the paternal and passively in the maternal genome, is a prominent feature of nuclear reprogramming; yet, this process poses a danger to a subset of methylated sequences that must be preserved for their germline to soma inheritance. Genomic imprinting and its importance were demonstrated three decades ago by a series of experiments generating non-viable mammalian uniparental embryos. Indeed, imprinted loci, gene clusters with parent-of-origin specific gene expression patterns, must retain their differential methylation status acquired during gametogenesis throughout embryogenesis and in adult tissues. It is just recently that the molecular players that protect/maintain imprinting marks during reprogramming in preimplantation embryos have been identified, in particular, an epigenetic modifier complex formed by ZFP57 and TRIM28/KAP1. The interaction of these and other molecules with the newly formed embryonic chromatin and imprinted genes is discussed and highlighted herein.     

Nuclear lamin antigen and messenger RNA expression in bovine in vitro produced and nuclear transfer embryos

The nuclear lamina is a complex meshwork of nuclear lamin filaments that lies on the interface of the nuclear envelope and chromatin and is important for cell maintenance, nucleoskeleton support, chromatin remodeling, and protein recruitment to the inner nucleolus. Protein and mRNA patterns for the major nuclear lamins were investigated in bovine in vitro fertilized (IVF) and nuclear transfer embryos. Expression of lamins A/C and B were examined in IVF bovine germinal vesicle (GV) oocytes, metaphase II oocytes, zygotes, 2-cell, 8-cell, 16-32-cell embryos, morulae, and blastocysts (n = 10). Lamin A/C was detected in 9/10 immature oocytes, 10/10 zygotes, 8/10 2-cell embryos, 4/10 morulae, 10/10 blastocysts but absent during the maternal embryonic transition. Lamin B was ubiquitously expressed during IVF preimplantation development but was only detected in 4/10 GV oocytes. Messenger RNA expression confirms that the major lamins, A/C and B1 are expressed throughout preimplantation development and transcribed by the embryo proper. Lamin A/C and B expression were observed (15 min, 30 min, 60 min, 120 min) following somatic cell nuclear transfer using adult fibroblasts and at the 2-cell, 8-cell, 16-32-cell, morula and blastocyst stage (n = 5). Altered expression levels and localization of nuclear lamins A/C and B was determined in nuclear transfer embryos during the first 2 hr post fusion, coincidental with only partial nuclear envelope breakdown as well as during the initial cleavage divisions, but was restored by the morula stage. This mechanical and molecular disruption of the nuclear lamina provides key evidence for incomplete nuclear remodeling and reprogramming following somatic cell nuclear transfer.     

Simultaneous Detection of FISH Signals and Bromo-Deoxyuridine Incorporation in Fixed Tissue Cultured Cells

FISH (Fluorescence in situ hybridization) is a powerful technique that detects and localises specific DNA sequences on metaphase chromosomes, interphase nuclei or chromatin fibres. When coupled to BrdU (5-Bromo 2-deoxy-uridine) labeling of newly replicated DNA, the replication properties of different DNA sequences can be analysed. However, the technique for the detection of BrdU incorporation is time consuming, and relies on acidic pH buffer treatments, that prevent use of pH sensitive fluorochromes such as FITC (Fluoro-isothiocianate) during FISH. In this work, we describe a simplified protocol that allows the simultaneous detection of FISH signals and BrdU incorporation. Since the technique does not involve paraformaldehyde for cell fixation, or formamide for denaturation of the target DNA and in post-hybridisation washes, it represents a safer alternative to classical FISH techniques.     

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specificationin mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).     

Estimates of mRNA abundance in the mouse blastocyst based on cDNA library analysis

Studies of gene expression during blastocyst formation in mouse preimplantation development have been limited by the amount of RNA available per embryo. Our present approach to this problem has been to construct a large, representative, blastocyst cDNA library in lambda gt11. Random hexadeoxynucleotides were used as primers with total blastocyst RNA serving as template. RNA collected from 4,100 32-64 cell embryos was used to generate a library with an initial size of 30 X 10(6) recombinants. By using clone frequency as a measure of relative mRNA abundance, our data support previous work on the relative and absolute amounts of actin, histone H2a, and intracisternal A particle. Furthermore, we provide estimates for the abundance of cytokeratin endo A, cytokeratin endo B, and beta-tubulin from clone frequency data. Insert sizes for isolated clones range from 200 bp to 3.6 kb with full-length or near-full-length insert sizes for selected clones, indicating that random primer methods generate cDNAs which can represent a significant portion of the mRNA. We have so far characterized products whose abundance is equal to or greater than 0.002% of total RNA. This library offers the potential for the analyses of presumptive regulatory gene products in the mouse preimplantation embryo which are represented as low abundance (less than 1% of mRNA) RNAs.