Specific gene expression during cell differentiation results from the concerted effects of intermingled factors: epigenetic modifications of DNA and histones, fixation of transcriptional factors, nuclear localization of genes, and the formation of higher-order chromatin structures. Indeed, over the past decade, the dynamic, temporal, and spatial organization of the eukaryotic cell nucleus has emerged as a central determinant of genome function [1–4].
When analyzing the correlation between nuclear organization and differentiation, early embryonic development offers a particularly interesting, although extremely complex, system. Upon fertilization, the highly specialized male and female gametes must be reprogrammed to form a totipotent embryo that will then differentiate and give rise to all the tissues of a new individual [5, 6]. In mammals, these events occur throughout the preimplantation period (in the female reproductive tract) and are thus accessible to detailed experimental investigations, especially in the mouse model. From large-scale transcriptomic analyses performed worldwide, it is now clear that this “reprogramming” process is dependent on finely tuned mechanisms of gene regulation . However, few researchers have analyzed structural and functional genome organization during early embryonic development [8–11].
Many studies focusing on epigenetic modifications have shown that, immediately after fertilization, both parental genomes undergo extensive remodeling during early cell cycles that is correlated with major modifications of gene expression . However, while parental genomes are first transcriptionally silenced in zygotes after fertilization, the embryonic genome is progressively turned on: in the mouse, a "minor activation" occurs at the end of the 1-cell stage , followed by a "major activation" at the 2-cell stage . This onset of embryonic gene expression (i.e. EGA, embryonic genome activation) is characterized by a rapid increase in the synthesis of transcripts . At the same time, transcription of ribosomal DNA (rDNA) is switched off in early mouse embryos and nucleoli are not present; instead, so-called nucleolar-precursor bodies (NPBs) are formed. The reinitiation of rDNA transcription occurs at the end of the 2-cell stage, at the surface of the NPBs . The first differentiation events take place later on with the physical and functional separation of the inner cell mass from the trophectoderm, at the blastocyst stage (day 3.5).
Parallel, large-scale mapping studies in somatic cells have shown that chromatin is not randomly distributed within nuclei but forms higher-order chromatin structures, some of which correlate with cell differentiation and gene activity [16–18]. For example, proximity to pericentromeric heterochromatin is generally associated with gene silencing [19, 20]. Centromeric and pericentromeric heterochromatic regions are highly important for chromosome stability and proper segregation . However, during interphase, these regions form higher-order chromatin structures – the so-called “chromocenter” clusters  – that act as transcriptionally repressive structures for genes spatially located in their vicinity [16, 23]. Similarly, it has been found that silencing of rDNA genes is tightly linked to heterochromatin formation .
When higher-order chromatin structures such as pericentromeric heterochromatin were first analyzed in the mouse, a specific nuclear architecture exclusive to the first embryonic cleavages was observed [8, 9]. Decondensation of pericentromeric heterochromatin seems to take place rapidly after fertilization, and it has been suggested that this maintains transcriptional silencing until EGA . Thereafter, reorganization of the centromeric and pericentromeric heterochromatin into “chromocenters” occurs concomitantly with the major phase of EGA [8–10]. In fact, interference with the reprogramming of the pericentromeric structures significantly alters development; it has been shown that disruption of chromocenters in mouse fertilized embryos results in developmental arrest [11, 25] and that cloned embryos produced by nuclear transfer often show aberrant nuclear architectures with remnants of somatic-like chromocenters, correlating with poor developmental rates [9, 26, 27].
Most of these results were acquired through the use of immuno-fluorescence and fluorescence in situ hybridization (FISH) to label compartments of interest in embryos. However, one important limitation of these studies is that the analysis of the corresponding fluorescent images is mostly visual and focused on large-scale nuclear movements, which are easier to evaluate. Genome wide approaches, especially chromosome conformation capture (3C), can provide more details to help decipher key nuclear events at the molecular level , but their use in embryos is limited due to the small size/number of the samples.
Fluorescent imaging offers us the advantage of following several structures within each embryo, thanks to high-resolution microscopy and the combination of several color channels. However, most analyses are done either in two dimensions or on z-stack sections/projections, and only rarely in three dimensions (3D) because they would be much more time-consuming. A promising approach to explore the embryonic nucleus in more detail is the use of computational imaging . At present, we are still at the very beginning of this approach, and the tools required to locate compartments of interest, to analyze their movements, and to measure physical distances still need improvement. Using this technique, however, Koehler and collaborators were recently able to describe, for the first time, 3D rearrangements of chromosome territories in preimplantation embryos . We similarly analyzed major 3D nuclear rearrangements of centromeric and pericentromeric heterochromatin in bovine and rabbit embryos with dedicated computational programs [30, 31].
To obtain a more complete understanding of the nuclear reorganization that takes place during the early developmental stages in mouse, we analyzed, in detail, centromeric and pericentromeric chromatin local reprogramming in preimplantation embryos with preserved 3D-shapes (from the 1-cell to blastocyst stage). We also developed new image analysis tools to quantify various nuclear parameters of the 3D-FISH images, i.e., the nuclear volume, the number of NPBs/nucleoli, the nuclear polarity, the number and shape of pericentromeric heterochromatin structures, and their proximity to NPBs/nucleoli.
Our results highlight differences in nuclear organization in paternal and maternal inherited genomes at the 1-cell stage. We also find that the reprogramming of the embryonic genome, which starts at the 2-cell stage, undergoes several abrupt changes during preimplantation development.