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Open Access Highly Accessed Research article

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

Tiphaine Aguirre-Lavin12, Pierre Adenot12, Amélie Bonnet-Garnier12, Gaétan Lehmann12, Renaud Fleurot12, Claire Boulesteix12, Pascale Debey12 and Nathalie Beaujean12*

Author Affiliations

1 INRA, UMR1198 Biologie du Développement et Reproduction, F-78350, Jouy-en-Josas, France

2 ENVA, F-94700, Maisons Alfort, France

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BMC Developmental Biology 2012, 12:30  doi:10.1186/1471-213X-12-30

Published: 24 October 2012

Abstract

Background

Embryonic 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).

Results

In 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.

Conclusions

The 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.

Keywords:
FISH; Heterochromatin; Centromeres; Telomeres; rDNA; Nucleolus; Nuclear organization; Embryo; Computational analysis