BACKGROUND
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
<cit> .
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
<cit> .
in mammals, these events occur throughout the preimplantation period  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
<cit> .
however, few researchers have analyzed structural and functional genome organization during early embryonic development
<cit> .
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
<cit> .
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
<cit> , followed by a "major activation" at the 2-cell stage
<cit> .
this onset of embryonic gene expression  is characterized by a rapid increase in the synthesis of transcripts
<cit> .
at the same time, transcription of ribosomal dna  is switched off in early mouse embryos and nucleoli are not present; instead, so-called nucleolar-precursor bodies  are formed.
the reinitiation of rdna transcription occurs at the end of the 2-cell stage, at the surface of the npbs
<cit> .
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 .
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
<cit> .
for example, proximity to pericentromeric heterochromatin is generally associated with gene silencing
<cit> .
centromeric and pericentromeric heterochromatic regions are highly important for chromosome stability and proper segregation
<cit> .
however, during interphase, these regions form higher-order chromatin structures – the so-called “chromocenter” clusters
<cit>  – that act as transcriptionally repressive structures for genes spatially located in their vicinity
<cit> .
similarly, it has been found that silencing of rdna genes is tightly linked to heterochromatin formation
<cit> .
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
<cit> .
decondensation of pericentromeric heterochromatin seems to take place rapidly after fertilization, and it has been suggested that this maintains transcriptional silencing until ega
<cit> .
thereafter, reorganization of the centromeric and pericentromeric heterochromatin into “chromocenters” occurs concomitantly with the major phase of ega
<cit> .
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
<cit>  and that cloned embryos produced by nuclear transfer often show aberrant nuclear architectures with remnants of somatic-like chromocenters, correlating with poor developmental rates
<cit> .
most of these results were acquired through the use of immuno-fluorescence and fluorescence in situ hybridization  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 , can provide more details to help decipher key nuclear events at the molecular level
<cit> , 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  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
<cit> .
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
<cit> .
we similarly analyzed major 3d nuclear rearrangements of centromeric and pericentromeric heterochromatin in bovine and rabbit embryos with dedicated computational programs
<cit> .
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 .
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.
RESULTS
unique nuclear organization of zygotes
we first analyzed the distribution of centromeric  and pericentromeric  heterochromatin in zygotes throughout the first cell cycle after fertilization .
at that stage, the parental genomes are separated in two haploid pronuclei  containing nonfunctional npbs, and zygotes can be classified in substages from pn <dig> to pn5
<cit> .
as previously described in the literature, we observed markedly different reorganizations within the male and female pronuclei from pn <dig> to pn <dig>  just after fertilization, pericentromeres organized rapidly around the npbs in the female pronucleus  whereas in the male pronucleus , they remained associated together in more or less unorganized masses located in the center .
remarkably, at pn <dig>  only ~3% of the npbs were not associated with pericentromeric signals in the fpn as opposed to almost 30% in the mpn .
we also noticed that the number of npbs, while decreasing with time in both pns, remained approximately twice as high in the mpn as compared to the fpn .
values with superscripts a and a’ are significantly different at p <  <dig> .
values with superscripts b and b’, and c and c’ are significantly different at p =  <dig> .
values with superscripts a and b are significantly different at p <  <dig> .
values with superscripts c are significantly different at p =  <dig> .
it was only in the late 1-cell stage  that pericentromeric heterochromatin adopted the same distribution in mpn and fpn, namely, more or less complete "shells" around the npbs, in which the minor satellite centromeric signals were embedded .
pericentromeric heterochromatin  was also found at the nuclear periphery , in association with centromeric spots.
pericentromeric heterochromatin formed other remarkable features such as “beaded” filaments extending from the nucleolar periphery towards the nuclear periphery .
in addition, as in earlier substages , the number of npbs remained lower in the fpns  than in the mpns  .
owing to the tight association we observed between pericentromeric heterochromatin and the npbs, we next analyzed the localization of rdna sequences also known to be structurally associated with npbs
<cit> .
for that purpose, we performed a dual fish with major satellite and rdna probes .
we found most of the rdna signals associated with pericentromeric signals at the periphery of the npbs or within the pericentromeric filaments .
we sometimes noticed rdna signals joining two npbs .
more surprisingly, we frequently observed rdna foci at the nuclear periphery, associated with pericentromeric signals .
in fact, ~80% of the pericentromeric signals at the nuclear periphery were flanked by rdna foci.
it should also be mentioned that none of the npbs devoid of pericentromeric signals were labeled with rdna, and ~30% of the pns contained npbs bearing pericentromeric signals but no rdna foci.
it thus appears that the strong association of pericentromeric heterochromatin with npbs is not restricted to chromosomes bearing rdna sequences, and that such chromosomes are not exclusively associated with npbs.
to gain deeper insight into chromatin higher-order organization within the pronuclei, we next analyzed the distribution of telomeres and performed triple-color fish with major satellite, minor satellite, and telomeric probes .
we could detect the same number of telomeric and centromeric spots in the fpn  and in the mpn  , which approached the expected numbers of  <dig> and  <dig>  respectively.
more interestingly, we found approximately half of the telomeres located around the npbs or associated with extra nucleolar pericentromeric signals  , together with an equivalent number of centromeres.
the second half appeared to be free in the nucleoplasm or close to the nuclear envelope .
at the end of the 1-cell stage, chromosomes condensed in both pns through a process equivalent to prometaphase
<cit> ; pericentromeres previously forming the peri-npb shell condensed with their corresponding centromeres and anchored the chromosomes to the npb, whereas the rest of the chromosomal scaffold extended outwards, like a “cartwheel”
<cit> .
one to three chromosomes seemed to escape from this radial organization and remained at the periphery of the cartwheel.
they most probably corresponded to the few centromeric/pericentromeric filaments and foci observed at the nuclear periphery from the pn <dig> to pn <dig> stages.
post-zygotic changes in nuclear organization
after the zygotic stage, the embryonic genome undergoes structural and functional changes.
for example, it is well-known that the compaction of pericentromeric heterochromatin that forms chromocenters starts at the 2-cell stage
<cit> .
however, few data exist on the global nuclear morphological changes occurring during pre-implantation development, up to the blastocyst stage.
we therefore performed systematic 3d-fish with minor and major satellite probes .
we analyzed embryos at various time-points during the 2-cell/4-cell/8-cell/16-cell/morula and blastocyst stages.
representative examples are shown in figure
<dig>
we observed that remodelling of the embryonic genome indeed started at the 2-cell stage.
at the beginning of the second cell cycle, the major satellites were essentially associated with the npbs, as in zygotes, forming either thick partial rims  or more spherical patches  .
centromeric spots were always associated with these rims and patches .
the remaining npbs  were free of any fish signal .
however, by the end of the second cell cycle, the percentage of npbs associated with spherical patches of pericentromeric heterochromatin increased , whereas npbs surrounded by rims tended to disappear  .
in these embryos, most of the rdna signals were located in close proximity to the npbs and the pericentromeric signals, as found in zygotes .
values with superscripts a and a’ are significantly different at p <  <dig> .
values with superscripts b and b’ differ at p =  <dig> .
remarkably, higher-order chromatin reorganization continued beyond the 2-cell stage.
new structures containing centromeric and pericentromeric heterochromatin appeared at the 4-cell stage, forming structures very similar to classical chromocenters, i.e., a compact mass of pericentromeric heterochromatin surrounded by individual centromeres .
during the same period, the number of nucleoli, which were now fully active
<cit> , underwent an abrupt decrease between early 4-cell  and late 4-cell stages .
finally, by the blastocyst stage, the overall nuclear organization was very similar to that of somatic cell nuclei in terms of nucleoli numbers and chromocenter organization
<cit> .
however, we scanned more than  <dig> embryos in total, making the image analysis tedious.
in these conditions, only the most obvious large-scale nuclear movements could be evaluated by visual analysis.
we therefore configured semi-automated image analysis tools particularly adapted to the size and geometry of the embryonic nuclei, describing quantitative morphometric features of the nuclei and the npbs/nucleoli.
we also analyzed, in detail, heterochromatin behavior in the context of such morphological changes.
morphometric features of nuclei and npbs/nucleoli
dna labeling was used to delineate the embryonic nuclei from the confocal 3d-stacks and to calculate nuclear volumes .
it should be mentioned that, for early stages, we distinguished early and late time points.
however, at later stages, cellular divisions were no longer synchronous and such an analysis could not be performed; we thus pooled the data within each stage.
figure
<dig> shows that the nuclear volume decreased progressively from the 2-cell stage  to the blastocyst stage  by a factor of  <dig>  with the most marked decrease occurring between the 2- and 4-cell stages .
we next performed a quantitative automated analysis of npb/nucleolus numbers and volumes.
as shown in figure
<dig> and table
<dig>  the number of npbs decreased slightly but significantly between the early and late 2-cell stage .
this decrease during the 2-cell stage was accompanied by a marked modification  in the distribution of npb volumes : the median value increased from  <dig>  μm <dig>  to  <dig>  μm <dig> .
interestingly, npbs associated with pericentromeric heterochromatin were larger than those not associated with pericentromeric heterochromatin, both at early  and late  stages.
at  <dig> cell, the changes in nucleolar number and size distribution are much smaller, suggesting that this fusion process is less prominent.
within each column, values with different subscripts are significantly different at p <  <dig> .
in the following stage, the number of npbs decreased drastically, as expected, from ~ <dig> npbs in early 4-cell  to ~ <dig> in late 4-cell embryos .
remarkably, the median value of the npb volume reached  <dig>  μm <dig> by the end of the 4-cell stage , suggesting that the number of npbs decreases via npb fusion.
controls performed on representative 2-cell and 4-cell embryos showed a slight divergence in the results obtained from manual counting of the npbs and those from the computerized image analysis, with 8% to 10% divergence in late 2-cell , early 2-cell , 4-cell  stages.
however, we observed that this difference was related to the smallest npbs only .
during later development, the number of nucleoli remained quite low , with a slight increase at the 8-cell stage .
however, we noticed that the total nucleolar volume relative to the nuclear volume decreased continuously from 15% at the 2-cell stage  to approximately 10% at the 16-cell  and 32-cell stages  .
pericentromeric heterochromatin structure and organization
one of the major events affecting centromeres and pericentromeres during preimplantation development is their relocation from the periphery of the npbs towards the nucleoplasm, where they form structures resembling the chromocenters in somatic mouse nuclei.
in order to gain deeper insight into this phenomenon, we analyzed the “roundness” of the pericentromeric 3d-fish signals: in brief, the surface of the object undergoing segmentation was divided by the surface of a sphere with an equivalent volume .
we could thereafter classify the pericentromeres either as “compact”, when their roundness was greater than  <dig>  and their size larger than  <dig>  μm <dig>  or “elongated”, when their roundness was less than  <dig>  and their size larger than  <dig>  μm <dig>  pericentromeric signals of less than  <dig>  μm <dig> were “not analyzed” ; these represented less than 5% of the total volume of pericentromeres at 2-cell and 4-cell stages and less than 10% in later stages.
figure
6a/a’/a” illustrates the segmentation and classification of the signals obtained using 3d-fish with pericentromeric probes on a 2-cell stage embryo.
we immediately noticed that pericentromeres partially surrounding npbs usually had a roundness that was less than  <dig> .
we therefore created another tool to analyze the relationship between elongated pericentromeres and npbs/nucleoli.
in brief, we measured the volume of pericentromeric signal interactions with npbs/nucleoli within a three-pixel distance from the nbps/nucleoli .
we then determined five categories: null or close for those without clear interactions versus low, medium, and strong for those with pericentromere and npb/nucleolus interactions.
figure
6b shows that the proportion of elongated pericentromeres with a strong npb/nucleolus interaction was higher in early 2-cell than in late 2-cell embryos.
it then decreased dramatically between the 2- and 4-cell stages, suggesting that the dissociation of pericentromeric heterochromatin from nbps/nucleoli begins at the 2-cell stage and finishes at the 4-cell stage.
interestingly, in some late 2-cell nuclei, we noticed apparently “compact” pericentromeres with a less intense “core”.
however, these pericentromeres were classified as “elongated” due to their crescent shape after segmentation .
we believe these pericentromeres represent intermediate configurations between elongated and compact heterochromatin structures.
both elongated and compact heterochromatin structures were present in 2-cell stage embryos.
figure
6d/d’ shows the percentage of each structure in nuclei of early 2-cell and late 2-cell stage embryos.
although these percentages vary from nucleus to nucleus, we could already observe important changes during the 2-cell stage: the percentage of compact pericentromeres was below 50% in the majority of the early 2-cell nuclei and above 50% in the majority of late 2-cell nuclei.
when we analyzed a larger number of nuclei from different experiments, we could still see that the percentage of “compact” pericentromeres increased between early and late 2-cell stages .
however, it clearly appears that this number increased more sharply between the 2-cell and 4-cell stages, when it reached 90%, a value that did not vary much thereafter .
maintenance of nuclear polarity of embryonic nuclei during preimplantation development
we next performed systematic 3d-fish with telomeric and pericentromeric probes on embryos from the 2-cell stage until blastocyst.
representative examples are shown in figure
<dig>  whereas we observed a peculiar radial distribution of the telomeres versus the centromeres/pericentromeres in the 1-cell stage, their spatial distribution at the 2-cell stage was completely different .
at the later stage, centromeres/pericentromeres and their corresponding telomeres were confined to one part of the nuclei , while the remaining telomeres were clustered in the other part .
this polarity, known as rabl-like configuration, has already been revealed in nuclei of 2-cell stage embryos via the staining of centromeres/pericentromeres
<cit>  and in some 8-cell embryos
<cit> .
it is believed to reflect the anaphase orientation of chromosomes and, as such, is usually lost in interphase, except in rare cases
<cit> .
strikingly, it seemed to be maintained in mouse preimplantation embryos during the 2-cell stage and in the following developmental stages .
as polarity is quite difficult to analyze visually in toto after the 4-cell stage, essentially because of the small nuclear sizes, we developed a quantitative method to evaluate polarity over a large collection of nuclei .
as shown in figure
<dig>  centromere distribution within the nuclei highlights the presence of polarity in all stages that were examined.
curiously, we noticed that this polarity is lower in the late 4-cell stage as compared to any other stage.
polarity increases again in early 8-cell embryos, then continuously decreases up to the blastocyst stage .
discussion
important structural remodeling and functional reprogramming affect the parental genomes during the critical preimplantation developmental period, which encompasses the transition from totipotency to differentiation.
in this study, we used fish with various genomic probes to analyze higher-order chromatin reorganization in detail on large numbers of mouse embryos with 3d preserved nuclei.
peculiar features of zygote nuclear organization
just after fertilization and during the 1-cell stage, the two parental genomes are still separated in two pronuclei.
this allows the observation of their different behaviors; for example, the differences in terms of epigenetic marks have already been well-documented
<cit> .
similarly, higher-order chromatin structures such as pericentromeric heterochromatin have also already been analyzed in mouse embryos
<cit> .
as described in the results section, we observed marked reorganizations within both pronuclei, male  and female , during the 1-cell stage.
just after fertilization, pericentromeres organize rapidly around the npbs in the fpn, but remain associated in more or less unorganized masses in the mpn.
through the detailed analysis of our 3d-fish images, we show here that paternal pericentromeric heterochromatin remains aggregated in a central mass up to the pn <dig> stage, and is only later dispersed to become associated with npbs.
this difference between the two parental genomes may be related to 1) the specific higher-order structure of sperm heterochromatin; 2) the progressive replacement of sperm protamines by histones; and/or 3) the specific epigenetic marks present only in male chromatin
<cit> .
regardless of their initial differences, by the end of the first cell cycle, maternal and paternal pericentromeric heterochromatin experience very similar decondensation states, together with a significant tendency to surround npbs.
this decondensation of pericentromeric heterochromatin takes place at the time of minor genome activation
<cit> , suggesting a direct functional link between the decondensation of pericentromeric heterochromatin and the transcriptional activation of the corresponding genomic sequences.
the highly decondensed state of pericentromeres at the 1-cell stage has also been observed by electron spectroscopic imaging
<cit> .
in our study, it is highlighted by the fact that “filaments” of pericentromeric signals could be observed escaping the periphery of the npbs towards that of the nucleus.
this result is quite surprising when compared to previous analyses performed by the immuno-staining of hp1β, the associated heterochromatin protein
<cit> .
we can infer from our results that hp1β is not associated to the totality of the pericentromeric heterochromatin and is absent from the radial filaments.
we believe this highly decondensed state of pericentromeres participates to the onset of pericentric satellites expression that starts in late 1-cell stage embryos
<cit> .
it is interesting to note that a similar “dispersion” of pericentromeric heterochromatin followed by a sequential reassembly was observed upon dedifferentiation-redifferentiation of arabidopsis leaf cells
<cit>  and in nuclear transfer experiments
<cit> .
taken together, these results suggest that this specific rearrangement of pericentromeric heterochromatin could be one of the features of totipotency.
importance of npbs/nucleoli in global nuclear organization
in the present study, we analyzed the distribution of centromeric/pericentromeric heterochromatin with respect to the nucleolar precursor bodies and nucleoli known to evolve rapidly throughout the first developmental stages.
in 1-cell and 2-cell stage embryos, we observed a tight association of this type of heterochromatin with npbs/nucleoli, as previously described
<cit> .
this tight association does not however hold for all chromosomes, since pericentromeric heterochromatin foci were also found at the nuclear periphery in interphasic 1-cell embryos  and escaping “peripheral” chromosomes are observed at condensation .
whether these are specific chromosomes remains unknown; this could be analyzed by chromosome territories painting.
it was, however, quite surprising to find that, whenever pericentromeres were located at the nuclear periphery, rdna signals were almost always associated with them.
this confirms that rdna genomic sequences are not automatically associated with npbs
<cit> .
it also suggests that, at least in early stages, npbs are not basic nucleolar precursors, but may have another role and/or function.
this hypothesis is in agreement with the fact that oocyte nucleolar components are necessary for the reassembly of newly formed npbs in both pronuclei after fertilization and for further embryonic development
<cit> .
however, the exact composition of these prominent compact fibrillar structures, which are present in fully grown oocytes and early embryos, is far from being completely deciphered.
different approaches have shown that they do not contain dna, but rather rna, nucleolar proteins , and non-nucleolar spliceosomal factors
<cit> .
it is only during the first half of the 2-cell stage that components of the rdna synthesis machinery are progressively assembled at the npb surface, where the first rrnas are synthesized at the mid/late-2-cell stage
<cit> .
remarkably, while a small but significant cell-cycle-dependent decrease in npb number is observed at 1-cell and 2-cell stages, the decrease is more drastic in 4-cell embryos and is accompanied by a large increase in the median npb volume.
this might reflect a rapid transition in the npbs’ function.
indeed, if the onset of rrna synthesis was previously precisely timed
<cit> , nothing is known concerning the dynamics of the other steps of rrna maturation and pre-ribosomal particles assembly.
from a more structural point of view, the fact that the decrease in npb number is associated with an increase in the median npb volume, without a significant reduction in the overall volume, suggests the existence of a fusion process in early embryos.
a similar fusion process could explain the slight decrease in npb number at late 1-cell, as suggested by the rdna bridges sometimes observed between  <dig> npbs .
remarkably, the increase of the npb volume stops at the 4-cell stage and is not observed anymore at the 8-cell stage.
this would fit with the fact that active polymerase i transcription and related processing machineries are functionally organized at the npb surface starting from the end of the 4-cell stage
<cit> .
this fusion process of the npb could reflect the entropy-driven nonspecific self-organizing forces  proposed by some authors to underlie the principles of nuclear organization
<cit> .
recent computer simulation of chromatin dynamics
<cit>  suggests that these “depletion-attraction” forces are sufficient to explain the position of chromocenters and nucleoli in interphasic arabidopsis nuclei.
structural features of the centromeres/pericentromeres in post-zygotic embryonic stages
because of the highly decondensed state of pericentromeric heterochromatin in zygotes, we were not able to segment the fish signals in these embryos with sufficient precision to perform further computational analysis.
on the other hand, as reorganization of the centromeric and pericentromeric heterochromatin into “chromocenters” occurs post-zygotically, in subsequent stages we were able to more precisely analyze heterochromatin reorganization as well as various nuclear parameters using the 3d-fish images.
unique image analysis tools developed for large objects such as individual embryos in toto were specifically adapted to analyze nuclear elements of highly different and complex sizes and shapes, especially the pericentromeric signals.
finally, as all the segmented signals/objects bore labels, we were able to analyze their relationships and measure interaction volumes.
thanks to these computational tools, we were able to analyze, for the first time, a large number of embryos  covering the whole preimplantation period.
these methods also allowed us to statistically document development-dependent modifications of embryonic genome organization.
in particular, we show here that nuclear polarity is conserved up to the 32-cell stage but decreases in blastocysts, as previously suggested by 2d-fish on centromeric repeats
<cit> .
unexpectedly, we also found that the 4-cell stage represents a major step in preimplantation development.
when we classified the pericentromeres as either “compact” or “elongated”, we observed that the proportion of elongated pericentromeres with a strong npb/nucleolus interaction was higher in the early 2-cell than in the late 2-cell stage.
this proportion then decreased dramatically between the 2- and 4-cell stages, while the percentage of “compact” pericentromeres increased drastically to reach 90%.
altogether, this suggests that dissociation of pericentromeric heterochromatin from nbps/nucleoli begins at the 2-cell stage but finishes at the 4-cell stage.
the factors or mechanisms that first favor pericentromeric/centromeric association to npbs and then initiate the formation of chromocenters remain largely unknown.
however, one such factor could be the hp1β protein.
in somatic cells, the presence of hp1β in fibrillarin-rich regions of nucleoli has already been reported
<cit> .
in mouse 1- and 2-cell embryos, we previously showed that fibrillarin is located at the npb surface
<cit>  and could therefore represent an anchoring protein for hp1β and pericentromeric heterochromatin.
indeed, in 1-cell stage embryos, hp1β accumulations are detectable in the fpn mainly around npbs and have been also detected, in lower amounts, in association with the paternal pericentromeres
<cit> .
this hypothesis is supported by recent data showing hp <dig> mislocalization, abnormal nuclear organization, and developmental arrest in h <dig>  k27r mutant embryos
<cit> .
modifications of epigenetic marks could also be involved in the reorganization of pericentromeric/centromeric chromatin.
complex and asymmetric histone/dna modifications occur continuously throughout early development in both parental genomes and may regulate the balance between pericentromeric “elongated” versus “compact” structures
<cit> .
finally, regarding other factors that might play a role in heterochromatin assembly and organization, it must be recalled that an unidentified rna is an integral component of pericentromeric heterochromatin in humans
<cit>  and is necessary for the accumulation of hp1α on pericentromeric heterochromatin
<cit> .
moreover, small centromeric rnas are involved in murine centromeric heterochromatin assembly
<cit> , and in mouse embryos, small rnas seem to participate in the formation of chromocenters as shown by the use of locked nucleic acid -dna gapmers to interfere with the transcription of major satellites in early embryos.
this induces developmental arrest before the completion of chromocenter formation
<cit> .
CONCLUSIONS
altogether, our results show that significant genome restructuring occurs during the entire preimplantation period.
just after fertilization, zygotes have a very peculiar nuclear organization with highly decondensed pericentromeric heterochromatin structures.
during the next cell cycle , nucleolar precursor bodies  and pericentromeric heterochromatin undergo important reorganization, as previously described in the literature.
however, thanks to new computational tools, we were able to analyze these elements for the first time in a large number of embryos, all the way up to the blastocyst stage.
we believe that these tools could now be used to enable detailed analyses of fluorescent 3d images in other models/organisms.
specifically, we consider computational imaging a promising approach to explore large-scale nuclear movements.
we also demonstrated that the 4-cell stage represents a major step in preimplantation development, especially with regards to pericentromeric structures.
pericentromeric structures may impact the regulation of developmental genes, particularly on heterochromatin-dependent gene silencing.
as in somatic cells, the role of these rearrangements during preimplantation development may be to bring different nuclear compartments  in close proximity in order to activate/repress specific genes yet to be identified.

 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. 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. in mammals, these events occur throughout the preimplantation period 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. 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 is characterized by a rapid increase in the synthesis of transcripts. at the same time, transcription of ribosomal dna is switched off in early mouse embryos and nucleoli are not present; instead, so-called nucleolar-precursor bodies 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. 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. for example, proximity to pericentromeric heterochromatin is generally associated with gene silencing. 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. 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. 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. 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 and that cloned embryos produced by nuclear transfer often show aberrant nuclear architectures with remnants of somatic-like chromocenters, correlating with poor developmental rates. most of these results were acquired through the use of immuno-fluorescence and fluorescence in situ hybridization 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, 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 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. 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. 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.  unique nuclear organization of zygotes we first analyzed the distribution of centromeric and pericentromeric heterochromatin in zygotes throughout the first cell cycle after fertilization. at that stage, the parental genomes are separated in two haploid pronuclei containing nonfunctional npbs, and zygotes can be classified in substages from pn to pn5. as previously described in the literature, we observed markedly different reorganizations within the male and female pronuclei from pn to pn just after fertilization, pericentromeres organized rapidly around the npbs in the female pronucleus whereas in the male pronucleus, they remained associated together in more or less unorganized masses located in the center. remarkably, at pn only ~3% of the npbs were not associated with pericentromeric signals in the fpn as opposed to almost 30% in the mpn. we also noticed that the number of npbs, while decreasing with time in both pns, remained approximately twice as high in the mpn as compared to the fpn. values with superscripts a and a’ are significantly different at p <. values with superscripts b and b’, and c and c’ are significantly different at p =. values with superscripts a and b are significantly different at p <. values with superscripts c are significantly different at p =. it was only in the late 1-cell stage that pericentromeric heterochromatin adopted the same distribution in mpn and fpn, namely, more or less complete "shells" around the npbs, in which the minor satellite centromeric signals were embedded. pericentromeric heterochromatin was also found at the nuclear periphery, in association with centromeric spots. pericentromeric heterochromatin formed other remarkable features such as “beaded” filaments extending from the nucleolar periphery towards the nuclear periphery. in addition, as in earlier substages, the number of npbs remained lower in the fpns than in the mpns. owing to the tight association we observed between pericentromeric heterochromatin and the npbs, we next analyzed the localization of rdna sequences also known to be structurally associated with npbs. for that purpose, we performed a dual fish with major satellite and rdna probes. we found most of the rdna signals associated with pericentromeric signals at the periphery of the npbs or within the pericentromeric filaments. we sometimes noticed rdna signals joining two npbs. more surprisingly, we frequently observed rdna foci at the nuclear periphery, associated with pericentromeric signals. in fact, ~80% of the pericentromeric signals at the nuclear periphery were flanked by rdna foci. it should also be mentioned that none of the npbs devoid of pericentromeric signals were labeled with rdna, and ~30% of the pns contained npbs bearing pericentromeric signals but no rdna foci. it thus appears that the strong association of pericentromeric heterochromatin with npbs is not restricted to chromosomes bearing rdna sequences, and that such chromosomes are not exclusively associated with npbs. to gain deeper insight into chromatin higher-order organization within the pronuclei, we next analyzed the distribution of telomeres and performed triple-color fish with major satellite, minor satellite, and telomeric probes. we could detect the same number of telomeric and centromeric spots in the fpn and in the mpn, which approached the expected numbers of and respectively. more interestingly, we found approximately half of the telomeres located around the npbs or associated with extra nucleolar pericentromeric signals, together with an equivalent number of centromeres. the second half appeared to be free in the nucleoplasm or close to the nuclear envelope. at the end of the 1-cell stage, chromosomes condensed in both pns through a process equivalent to prometaphase; pericentromeres previously forming the peri-npb shell condensed with their corresponding centromeres and anchored the chromosomes to the npb, whereas the rest of the chromosomal scaffold extended outwards, like a “cartwheel”. one to three chromosomes seemed to escape from this radial organization and remained at the periphery of the cartwheel. they most probably corresponded to the few centromeric/pericentromeric filaments and foci observed at the nuclear periphery from the pn to pn stages. post-zygotic changes in nuclear organization after the zygotic stage, the embryonic genome undergoes structural and functional changes. for example, it is well-known that the compaction of pericentromeric heterochromatin that forms chromocenters starts at the 2-cell stage. however, few data exist on the global nuclear morphological changes occurring during pre-implantation development, up to the blastocyst stage. we therefore performed systematic 3d-fish with minor and major satellite probes. we analyzed embryos at various time-points during the 2-cell/4-cell/8-cell/16-cell/morula and blastocyst stages. representative examples are shown in figure we observed that remodelling of the embryonic genome indeed started at the 2-cell stage. at the beginning of the second cell cycle, the major satellites were essentially associated with the npbs, as in zygotes, forming either thick partial rims or more spherical patches. centromeric spots were always associated with these rims and patches. the remaining npbs were free of any fish signal. however, by the end of the second cell cycle, the percentage of npbs associated with spherical patches of pericentromeric heterochromatin increased, whereas npbs surrounded by rims tended to disappear. in these embryos, most of the rdna signals were located in close proximity to the npbs and the pericentromeric signals, as found in zygotes. values with superscripts a and a’ are significantly different at p <. values with superscripts b and b’ differ at p =. remarkably, higher-order chromatin reorganization continued beyond the 2-cell stage. new structures containing centromeric and pericentromeric heterochromatin appeared at the 4-cell stage, forming structures very similar to classical chromocenters, i.e., a compact mass of pericentromeric heterochromatin surrounded by individual centromeres. during the same period, the number of nucleoli, which were now fully active, underwent an abrupt decrease between early 4-cell and late 4-cell stages. finally, by the blastocyst stage, the overall nuclear organization was very similar to that of somatic cell nuclei in terms of nucleoli numbers and chromocenter organization. however, we scanned more than embryos in total, making the image analysis tedious. in these conditions, only the most obvious large-scale nuclear movements could be evaluated by visual analysis. we therefore configured semi-automated image analysis tools particularly adapted to the size and geometry of the embryonic nuclei, describing quantitative morphometric features of the nuclei and the npbs/nucleoli. we also analyzed, in detail, heterochromatin behavior in the context of such morphological changes. morphometric features of nuclei and npbs/nucleoli dna labeling was used to delineate the embryonic nuclei from the confocal 3d-stacks and to calculate nuclear volumes. it should be mentioned that, for early stages, we distinguished early and late time points. however, at later stages, cellular divisions were no longer synchronous and such an analysis could not be performed; we thus pooled the data within each stage. figure shows that the nuclear volume decreased progressively from the 2-cell stage to the blastocyst stage by a factor of with the most marked decrease occurring between the 2- and 4-cell stages. we next performed a quantitative automated analysis of npb/nucleolus numbers and volumes. as shown in figure and table the number of npbs decreased slightly but significantly between the early and late 2-cell stage. this decrease during the 2-cell stage was accompanied by a marked modification in the distribution of npb volumes: the median value increased from μm to μm. interestingly, npbs associated with pericentromeric heterochromatin were larger than those not associated with pericentromeric heterochromatin, both at early and late stages. at cell, the changes in nucleolar number and size distribution are much smaller, suggesting that this fusion process is less prominent. within each column, values with different subscripts are significantly different at p <. in the following stage, the number of npbs decreased drastically, as expected, from ~ npbs in early 4-cell to ~ in late 4-cell embryos. remarkably, the median value of the npb volume reached μm by the end of the 4-cell stage, suggesting that the number of npbs decreases via npb fusion. controls performed on representative 2-cell and 4-cell embryos showed a slight divergence in the results obtained from manual counting of the npbs and those from the computerized image analysis, with 8% to 10% divergence in late 2-cell, early 2-cell, 4-cell stages. however, we observed that this difference was related to the smallest npbs only. during later development, the number of nucleoli remained quite low, with a slight increase at the 8-cell stage. however, we noticed that the total nucleolar volume relative to the nuclear volume decreased continuously from 15% at the 2-cell stage to approximately 10% at the 16-cell and 32-cell stages. pericentromeric heterochromatin structure and organization one of the major events affecting centromeres and pericentromeres during preimplantation development is their relocation from the periphery of the npbs towards the nucleoplasm, where they form structures resembling the chromocenters in somatic mouse nuclei. in order to gain deeper insight into this phenomenon, we analyzed the “roundness” of the pericentromeric 3d-fish signals: in brief, the surface of the object undergoing segmentation was divided by the surface of a sphere with an equivalent volume. we could thereafter classify the pericentromeres either as “compact”, when their roundness was greater than and their size larger than μm or “elongated”, when their roundness was less than and their size larger than μm pericentromeric signals of less than μm were “not analyzed”; these represented less than 5% of the total volume of pericentromeres at 2-cell and 4-cell stages and less than 10% in later stages. figure 6a/a’/a” illustrates the segmentation and classification of the signals obtained using 3d-fish with pericentromeric probes on a 2-cell stage embryo. we immediately noticed that pericentromeres partially surrounding npbs usually had a roundness that was less than. we therefore created another tool to analyze the relationship between elongated pericentromeres and npbs/nucleoli. in brief, we measured the volume of pericentromeric signal interactions with npbs/nucleoli within a three-pixel distance from the nbps/nucleoli. we then determined five categories: null or close for those without clear interactions versus low, medium, and strong for those with pericentromere and npb/nucleolus interactions. figure 6b shows that the proportion of elongated pericentromeres with a strong npb/nucleolus interaction was higher in early 2-cell than in late 2-cell embryos. it then decreased dramatically between the 2- and 4-cell stages, suggesting that the dissociation of pericentromeric heterochromatin from nbps/nucleoli begins at the 2-cell stage and finishes at the 4-cell stage. interestingly, in some late 2-cell nuclei, we noticed apparently “compact” pericentromeres with a less intense “core”. however, these pericentromeres were classified as “elongated” due to their crescent shape after segmentation. we believe these pericentromeres represent intermediate configurations between elongated and compact heterochromatin structures. both elongated and compact heterochromatin structures were present in 2-cell stage embryos. figure 6d/d’ shows the percentage of each structure in nuclei of early 2-cell and late 2-cell stage embryos. although these percentages vary from nucleus to nucleus, we could already observe important changes during the 2-cell stage: the percentage of compact pericentromeres was below 50% in the majority of the early 2-cell nuclei and above 50% in the majority of late 2-cell nuclei. when we analyzed a larger number of nuclei from different experiments, we could still see that the percentage of “compact” pericentromeres increased between early and late 2-cell stages. however, it clearly appears that this number increased more sharply between the 2-cell and 4-cell stages, when it reached 90%, a value that did not vary much thereafter. maintenance of nuclear polarity of embryonic nuclei during preimplantation development we next performed systematic 3d-fish with telomeric and pericentromeric probes on embryos from the 2-cell stage until blastocyst. representative examples are shown in figure whereas we observed a peculiar radial distribution of the telomeres versus the centromeres/pericentromeres in the 1-cell stage, their spatial distribution at the 2-cell stage was completely different. at the later stage, centromeres/pericentromeres and their corresponding telomeres were confined to one part of the nuclei, while the remaining telomeres were clustered in the other part. this polarity, known as rabl-like configuration, has already been revealed in nuclei of 2-cell stage embryos via the staining of centromeres/pericentromeres and in some 8-cell embryos. it is believed to reflect the anaphase orientation of chromosomes and, as such, is usually lost in interphase, except in rare cases. strikingly, it seemed to be maintained in mouse preimplantation embryos during the 2-cell stage and in the following developmental stages. as polarity is quite difficult to analyze visually in toto after the 4-cell stage, essentially because of the small nuclear sizes, we developed a quantitative method to evaluate polarity over a large collection of nuclei. as shown in figure centromere distribution within the nuclei highlights the presence of polarity in all stages that were examined. curiously, we noticed that this polarity is lower in the late 4-cell stage as compared to any other stage. polarity increases again in early 8-cell embryos, then continuously decreases up to the blastocyst stage. discussion important structural remodeling and functional reprogramming affect the parental genomes during the critical preimplantation developmental period, which encompasses the transition from totipotency to differentiation. in this study, we used fish with various genomic probes to analyze higher-order chromatin reorganization in detail on large numbers of mouse embryos with 3d preserved nuclei. peculiar features of zygote nuclear organization just after fertilization and during the 1-cell stage, the two parental genomes are still separated in two pronuclei. this allows the observation of their different behaviors; for example, the differences in terms of epigenetic marks have already been well-documented. similarly, higher-order chromatin structures such as pericentromeric heterochromatin have also already been analyzed in mouse embryos. as described in the results section, we observed marked reorganizations within both pronuclei, male and female, during the 1-cell stage. just after fertilization, pericentromeres organize rapidly around the npbs in the fpn, but remain associated in more or less unorganized masses in the mpn. through the detailed analysis of our 3d-fish images, we show here that paternal pericentromeric heterochromatin remains aggregated in a central mass up to the pn stage, and is only later dispersed to become associated with npbs. this difference between the two parental genomes may be related to 1) the specific higher-order structure of sperm heterochromatin; 2) the progressive replacement of sperm protamines by histones; and/or 3) the specific epigenetic marks present only in male chromatin. regardless of their initial differences, by the end of the first cell cycle, maternal and paternal pericentromeric heterochromatin experience very similar decondensation states, together with a significant tendency to surround npbs. this decondensation of pericentromeric heterochromatin takes place at the time of minor genome activation, suggesting a direct functional link between the decondensation of pericentromeric heterochromatin and the transcriptional activation of the corresponding genomic sequences. the highly decondensed state of pericentromeres at the 1-cell stage has also been observed by electron spectroscopic imaging. in our study, it is highlighted by the fact that “filaments” of pericentromeric signals could be observed escaping the periphery of the npbs towards that of the nucleus. this result is quite surprising when compared to previous analyses performed by the immuno-staining of hp1β, the associated heterochromatin protein. we can infer from our results that hp1β is not associated to the totality of the pericentromeric heterochromatin and is absent from the radial filaments. we believe this highly decondensed state of pericentromeres participates to the onset of pericentric satellites expression that starts in late 1-cell stage embryos. it is interesting to note that a similar “dispersion” of pericentromeric heterochromatin followed by a sequential reassembly was observed upon dedifferentiation-redifferentiation of arabidopsis leaf cells and in nuclear transfer experiments. taken together, these results suggest that this specific rearrangement of pericentromeric heterochromatin could be one of the features of totipotency. importance of npbs/nucleoli in global nuclear organization in the present study, we analyzed the distribution of centromeric/pericentromeric heterochromatin with respect to the nucleolar precursor bodies and nucleoli known to evolve rapidly throughout the first developmental stages. in 1-cell and 2-cell stage embryos, we observed a tight association of this type of heterochromatin with npbs/nucleoli, as previously described. this tight association does not however hold for all chromosomes, since pericentromeric heterochromatin foci were also found at the nuclear periphery in interphasic 1-cell embryos and escaping “peripheral” chromosomes are observed at condensation. whether these are specific chromosomes remains unknown; this could be analyzed by chromosome territories painting. it was, however, quite surprising to find that, whenever pericentromeres were located at the nuclear periphery, rdna signals were almost always associated with them. this confirms that rdna genomic sequences are not automatically associated with npbs. it also suggests that, at least in early stages, npbs are not basic nucleolar precursors, but may have another role and/or function. this hypothesis is in agreement with the fact that oocyte nucleolar components are necessary for the reassembly of newly formed npbs in both pronuclei after fertilization and for further embryonic development. however, the exact composition of these prominent compact fibrillar structures, which are present in fully grown oocytes and early embryos, is far from being completely deciphered. different approaches have shown that they do not contain dna, but rather rna, nucleolar proteins, and non-nucleolar spliceosomal factors. it is only during the first half of the 2-cell stage that components of the rdna synthesis machinery are progressively assembled at the npb surface, where the first rrnas are synthesized at the mid/late-2-cell stage. remarkably, while a small but significant cell-cycle-dependent decrease in npb number is observed at 1-cell and 2-cell stages, the decrease is more drastic in 4-cell embryos and is accompanied by a large increase in the median npb volume. this might reflect a rapid transition in the npbs’ function. indeed, if the onset of rrna synthesis was previously precisely timed, nothing is known concerning the dynamics of the other steps of rrna maturation and pre-ribosomal particles assembly. from a more structural point of view, the fact that the decrease in npb number is associated with an increase in the median npb volume, without a significant reduction in the overall volume, suggests the existence of a fusion process in early embryos. a similar fusion process could explain the slight decrease in npb number at late 1-cell, as suggested by the rdna bridges sometimes observed between npbs. remarkably, the increase of the npb volume stops at the 4-cell stage and is not observed anymore at the 8-cell stage. this would fit with the fact that active polymerase i transcription and related processing machineries are functionally organized at the npb surface starting from the end of the 4-cell stage. this fusion process of the npb could reflect the entropy-driven nonspecific self-organizing forces proposed by some authors to underlie the principles of nuclear organization. recent computer simulation of chromatin dynamics suggests that these “depletion-attraction” forces are sufficient to explain the position of chromocenters and nucleoli in interphasic arabidopsis nuclei. structural features of the centromeres/pericentromeres in post-zygotic embryonic stages because of the highly decondensed state of pericentromeric heterochromatin in zygotes, we were not able to segment the fish signals in these embryos with sufficient precision to perform further computational analysis. on the other hand, as reorganization of the centromeric and pericentromeric heterochromatin into “chromocenters” occurs post-zygotically, in subsequent stages we were able to more precisely analyze heterochromatin reorganization as well as various nuclear parameters using the 3d-fish images. unique image analysis tools developed for large objects such as individual embryos in toto were specifically adapted to analyze nuclear elements of highly different and complex sizes and shapes, especially the pericentromeric signals. finally, as all the segmented signals/objects bore labels, we were able to analyze their relationships and measure interaction volumes. thanks to these computational tools, we were able to analyze, for the first time, a large number of embryos covering the whole preimplantation period. these methods also allowed us to statistically document development-dependent modifications of embryonic genome organization. in particular, we show here that nuclear polarity is conserved up to the 32-cell stage but decreases in blastocysts, as previously suggested by 2d-fish on centromeric repeats. unexpectedly, we also found that the 4-cell stage represents a major step in preimplantation development. when we classified the pericentromeres as either “compact” or “elongated”, we observed that the proportion of elongated pericentromeres with a strong npb/nucleolus interaction was higher in the early 2-cell than in the late 2-cell stage. this proportion then decreased dramatically between the 2- and 4-cell stages, while the percentage of “compact” pericentromeres increased drastically to reach 90%. altogether, this suggests that dissociation of pericentromeric heterochromatin from nbps/nucleoli begins at the 2-cell stage but finishes at the 4-cell stage. the factors or mechanisms that first favor pericentromeric/centromeric association to npbs and then initiate the formation of chromocenters remain largely unknown. however, one such factor could be the hp1β protein. in somatic cells, the presence of hp1β in fibrillarin-rich regions of nucleoli has already been reported. in mouse 1- and 2-cell embryos, we previously showed that fibrillarin is located at the npb surface and could therefore represent an anchoring protein for hp1β and pericentromeric heterochromatin. indeed, in 1-cell stage embryos, hp1β accumulations are detectable in the fpn mainly around npbs and have been also detected, in lower amounts, in association with the paternal pericentromeres. this hypothesis is supported by recent data showing hp mislocalization, abnormal nuclear organization, and developmental arrest in h k27r mutant embryos. modifications of epigenetic marks could also be involved in the reorganization of pericentromeric/centromeric chromatin. complex and asymmetric histone/dna modifications occur continuously throughout early development in both parental genomes and may regulate the balance between pericentromeric “elongated” versus “compact” structures. finally, regarding other factors that might play a role in heterochromatin assembly and organization, it must be recalled that an unidentified rna is an integral component of pericentromeric heterochromatin in humans and is necessary for the accumulation of hp1α on pericentromeric heterochromatin. moreover, small centromeric rnas are involved in murine centromeric heterochromatin assembly, and in mouse embryos, small rnas seem to participate in the formation of chromocenters as shown by the use of locked nucleic acid -dna gapmers to interfere with the transcription of major satellites in early embryos. this induces developmental arrest before the completion of chromocenter formation.  altogether, our results show that significant genome restructuring occurs during the entire preimplantation period. just after fertilization, zygotes have a very peculiar nuclear organization with highly decondensed pericentromeric heterochromatin structures. during the next cell cycle, nucleolar precursor bodies and pericentromeric heterochromatin undergo important reorganization, as previously described in the literature. however, thanks to new computational tools, we were able to analyze these elements for the first time in a large number of embryos, all the way up to the blastocyst stage. we believe that these tools could now be used to enable detailed analyses of fluorescent 3d images in other models/organisms. specifically, we consider computational imaging a promising approach to explore large-scale nuclear movements. we also demonstrated that the 4-cell stage represents a major step in preimplantation development, especially with regards to pericentromeric structures. pericentromeric structures may impact the regulation of developmental genes, particularly on heterochromatin-dependent gene silencing. as in somatic cells, the role of these rearrangements during preimplantation development may be to bring different nuclear compartments in close proximity in order to activate/repress specific genes yet to be identified. 
the results highlight differences in nuclear organization between the two parental inherited genomes at the 1-cell stage, i.e. 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. early embryonic development thus offers a particularly interesting system in which to analyze functional nuclear organization. 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. embryonic development proceeds through finely tuned reprogramming of the parental genomes to form a totipotent embryo. 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. in the present study, we used dna fluorescent in situ hybridization to localize centromeric, pericentromeric, and telomeric genomic sequences throughout the preimplantation period in naturally fertilized mouse embryos. 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. 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.
 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.  in the present study, we used dna fluorescent in situ hybridization to localize centromeric, pericentromeric, and telomeric genomic sequences throughout the preimplantation period in naturally fertilized mouse embryos. 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.  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. fishheterochromatincentromerestelomeresrdnanucleolusnuclear organizationembryocomputational analysis 