3D-FISH analysis of embryonic nuclei in mouse highlights several abrupt changes of nuclear organization during preimplantation development

Abstract

BackgroundEmbryonic development proceeds through finely tuned reprogramming of the parental genomes to form a totipotent embryo. Cells within this embryo will then differentiate and give rise to all the tissues of a new individual. Early embryonic development thus offers a particularly interesting system in which to analyze functional nuclear organization. When the organization of higher-order chromatin structures, such as pericentromeric heterochromatin, was first analyzed in mouse embryos, specific nuclear rearrangements were observed that correlated with embryonic genome activation at the 2-cell stage. However, most existing analyses have been conducted by visual observation of fluorescent images, in two dimensions or on z-stack sections/projections, but only rarely in three dimensions (3D).ResultsIn the present study, we used DNA fluorescent in situ hybridization (FISH) to localize centromeric (minor satellites), pericentromeric (major satellites), and telomeric genomic sequences throughout the preimplantation period in naturally fertilized mouse embryos (from the 1-cell to blastocyst stage). Their distribution was then analyzed in 3D on confocal image stacks, focusing on the nucleolar precursor bodies and nucleoli known to evolve rapidly throughout the first developmental stages. We used computational imaging to quantify various nuclear parameters in the 3D-FISH images, to analyze the organization of compartments of interest, and to measure physical distances between these compartments.ConclusionsThe results highlight differences in nuclear organization between the two parental inherited genomes at the 1-cell stage, i.e. just after fertilization. We also found that the reprogramming of the embryonic genome, which starts at the 2-cell stage, undergoes other remarkable changes during preimplantation development, particularly at the 4-cell stage.

Building up the nucleus: nuclear organization in the establishment of totipotency and pluripotency during mammalian development

“…In the maternal pronucleus, the pericentromeres occupy the surface of the NPB already from a few hours after pronuclear formation. In contrast, the pericentromeric repeats in the male pronucleus first cluster in an unorganized mass at the center of the pronucleus and restructure around the surface of the NPBs later compared with the maternal pronucleus (Aguirre-Lavin et al 2012). The chromatin structure of mature sperm provides a plausible explanation for this asymmetry, as pericentromeric domains are found in a single cluster in the center of the sperm nucleus (Haaf and Ward 1995).…”

“…Recently, rRNA transcription has been detected at the NPB as early as the zygote (Lin et al 2014), contrary to previous data (Zatsepina et al 2003), which suggested that rDNA transcription commences only at the late two-cell stage. The number of NPBs decreases during the cell cycle and over the progression from the zygote to the eight-cell stage, and the conventional tripartite morphology of somatic-type nucleoli gradually starts to appear forming around the NPB as a core (Flechon and Kopecny 1998;Aguirre-Lavin et al 2012). Importantly, within 3 h after SCNT, NPBs morphologically identical to those in fertilized embryos form, and their number correlates with developmental potential, raising the possibility that NPB formation might be a critical component of reprogramming to the totipotent state (Martin et al 2006b).…”

Positional changes of a pluripotency marker gene during structural reorganization of fibroblast nuclei in cloned early bovine embryos

“…The time-course of nuclear remodeling events differed during the first blastomere cleavage events, but IVF and cloned embryos achieved the same distinct nuclear organization in the majority of embryos at the 8-cell stage. Aguirre-Lavin et al 5 combined 3D-FISH, 3D-CLSM and quantitative 3D image analyses to study nuclear arrangements of centromeric (minor satellites), pericentromeric (major satellites), and telomeric genomic sequences, as well as nucleolar precursor bodies and nucleoli in naturally fertilized mouse embryos from the zygote to blastocyst stage. Remarkable changes of nucleolar precursor bodies and pericentromeric heterochromatin were noted during the transition from the mouse 2-cell to the 4-cell stage.…”

“…5,6,10 For this purpose, one has to take into account that mammalian species differ in the timing of major embryonic gene activation. 2 In rabbit embryos this event apparently starts, like in bovine embryos, at the 8-cell stage.…”

“…A number of groups, including ours, have made strong efforts to overcome the methodological obstacles preventing the detailed analysis of nuclear architecture in space and time during early mammalian development. [5][6][7][8][9][10][11] It has been reported that nuclear positions of genes, chromatin domains and even entire chromosome territories can change during development and cell differentiation. 8,[12][13][14] As an alternative to movements of entire chromosome territories, it was suggested that they may occupy the same nuclear place before and after activation of genes carried by them, whereas giant chromatin loops may form upon transcriptional activation and move such genes far away from their carrier chromosome territory into a nuclear environment favorable for their transcriptional activation and/or the maintenance of transcription.…”