Nuclear reorganisation in mouse pre-implantation embryos

Dynamics of nuclear re-organisation associated with genome reprogramming in mouse pre-implantation embryos

Jane Deuve, Agnès Dubois, Sara Merzouk, Julie Prudhomme, Céline Morey

Cell pluripotency and genome plasticity at pre-implantation stages are necessary to ensure a proper establishment of the various cell lineages of the embryo. Epigenetic marks such as specific chromatin structures or a specific organisation of the chromatin fibres within the cell nucleus might underlie this plasticity. To characterize these marks, we study the X-chromosome inactivation process, which is considered as a paradigm of large scale epigenetic reprogramming.

Figure 1. X-chromosome inactivation during pre-implantation development.
The mouse morula shows an imprinted inactivation of the paternal X (Xp) (Okamoto et al. 2004). In the early blastocyst - a time when differentiation of the first cell lineages occurs - this imprinted X-inactivation is conserved in extra-embryonic derivatives (primitive endoderm and trophectoderm) but is reverted in the inner cell mass where the random inactivation of the maternal (Xm) or the paternal X occurs (Okamoto et al., 2004; Mak et al., 2004). Afterwards, the inactive state is extremely stable and inherited during cell cycle. Reversion of this random X-inactivation only occurs in primordial germ cells. In the laboratory, we work on cell lines derived from the three lineages of the blastocyst (ES, TS and XEN cells (Kunath et al. 2005)) as well as directly on developing embryos.

We ask: what are the factors or epigenetic marks ensuring a stable inactive state; whether these marks are uniformly distributed along the inactive X-chromosome and what ensures X chromosome plasticity in pre-implantation embryos? To do this, we are bringing together a cell biology analysis of X chromosome nuclear organisation using 3D fluorescence in situ hybridisation (FISH) and a biochemical study of chromatin structure on high-resolution microarrays (ChIP on chip + ChIPSeq).

Figure 2. Nuclear movements and association with nuclear compartments.
Gene organisation within the nucleus is not random. Active transcription units tend to loop out from their chromosome territory (left panel) to be recruited to or excluded from constitutive nuclear compartments (examples of nuclear compartments on middle panel; example of gene recruitment to transcription factories on the right panel). These nuclear movements are thought to exert a crucial function in epigenetic regulation of gene expression.

We are also interested in the relationship between the initial lineage commitments and changes in X-inactivation states, i.e. how the different forms of X-inactivation (imprinted, random and Xp reactivation) are established at the morula-blastocyst transition; what are the molecular triggers ensuring these changes? For these studies we make use of mouse models available in the laboratory and of genetically engineered cell lines where lineage commitment is controlled.

Figure 3. Relationship between lineage commitment and X-inactivation.
The left panel shows that X-inactivation (identified by the presence of a Xist domain) is induced only in cells which have lost the pluripotency marker Nanog (Navarro et al., 2008). Trophectoderm cells can be obtained by inducible overexpression of Cdx2 in ES cells (Niwa et al., 2005) (middle panel). In order to monitor X-inactivation in vivo in each cell layer, we use an Xp-linked GFP transgene (Hadjantonakis et al., 1998).

Dynamics of nuclear re-organisation associated with genome reprogramming in mouse pre-implantation embryos Jane Deuve, Agnès Dubois, Sara Merzouk, Julie Prudhomme, Céline Morey
Cell pluripotency and genome plasticity at pre-implantation stages are necessary to ensure a proper establishment of the various cell lineages of the embryo. Epigenetic marks such as specific chromatin structures or a specific organisation of the chromatin fibres within the cell nucleus might underlie this plasticity. To characterize these marks, we study the X-chromosome inactivation process, which is considered as a paradigm of large scale epigenetic reprogramming.
	Figure 1. X-chromosome inactivation during pre-implantation development. The mouse morula shows an imprinted inactivation of the paternal X (Xp) (Okamoto et al. 2004). In the early blastocyst - a time when differentiation of the first cell lineages occurs - this imprinted X-inactivation is conserved in extra-embryonic derivatives (primitive endoderm and trophectoderm) but is reverted in the inner cell mass where the random inactivation of the maternal (Xm) or the paternal X occurs (Okamoto et al., 2004; Mak et al., 2004). Afterwards, the inactive state is extremely stable and inherited during cell cycle. Reversion of this random X-inactivation only occurs in primordial germ cells. In the laboratory, we work on cell lines derived from the three lineages of the blastocyst (ES, TS and XEN cells (Kunath et al. 2005)) as well as directly on developing embryos.
We ask: what are the factors or epigenetic marks ensuring a stable inactive state; whether these marks are uniformly distributed along the inactive X-chromosome and what ensures X chromosome plasticity in pre-implantation embryos? To do this, we are bringing together a cell biology analysis of X chromosome nuclear organisation using 3D fluorescence in situ hybridisation (FISH) and a biochemical study of chromatin structure on high-resolution microarrays (ChIP on chip + ChIPSeq).
	Figure 2. Nuclear movements and association with nuclear compartments. Gene organisation within the nucleus is not random. Active transcription units tend to loop out from their chromosome territory (left panel) to be recruited to or excluded from constitutive nuclear compartments (examples of nuclear compartments on middle panel; example of gene recruitment to transcription factories on the right panel). These nuclear movements are thought to exert a crucial function in epigenetic regulation of gene expression.
We are also interested in the relationship between the initial lineage commitments and changes in X-inactivation states, i.e. how the different forms of X-inactivation (imprinted, random and Xp reactivation) are established at the morula-blastocyst transition; what are the molecular triggers ensuring these changes? For these studies we make use of mouse models available in the laboratory and of genetically engineered cell lines where lineage commitment is controlled.
	Figure 3. Relationship between lineage commitment and X-inactivation. The left panel shows that X-inactivation (identified by the presence of a Xist domain) is induced only in cells which have lost the pluripotency marker Nanog (Navarro et al., 2008). Trophectoderm cells can be obtained by inducible overexpression of Cdx2 in ES cells (Niwa et al., 2005) (middle panel). In order to monitor X-inactivation in vivo in each cell layer, we use an Xp-linked GFP transgene (Hadjantonakis et al., 1998).