1. Volume 4, Issue 6, Pages 1156-1167 (September 2013)
Heterochromatin Reorganization during Early Mouse Development Requires a Single- Stranded Noncoding Transcript 
Miguel Casanova, Michał Pasternak, Fatima El Marjou, Patricia Le Baccon, Aline V. Probst, Geneviève Almouzni 
Cell Reports 
Volume 4, Issue 6, Pages (September 2013)
DOI: /j.celrep
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2. Cell Reports 2013 4, 1156-1167DOI: (10.1016/j.celrep.2013.08.015)
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3. Figure 1
Major Satellites Are Replicated during Mid–Late S Phase of the Two-Cell Embryo and Show Different Parental Replication Dynamics
(A) Shortly after completion of the first cleavage, embryos were pulsed every hour from 30 to 41 hr phCG by culturing in the presence of EdU for 1 hr. Immediately after the pulse, embryos were collected and processed for EdU detection together with either DNA FISH for major satellites or immunostaining for H3K9m3 or Ring1B. The graph represents the percentage of nuclei in which major satellites are labeled with EdU at different time frames within S phase of the two-cell stage.
(B) Two-cell embryos were pulsed with EdU at different time points during S phase. Representative embryos stained for H3K9me3 (red), enriched at the maternally derived genome and Ring1B (green), enriched at the paternally derived genome, followed by EdU click-iT (blue) are depicted. DNA is counterstained with DAPI (gray). Scale bar represents 5 μm. The graph represents the replication timing of paternal and maternal heterochromatin. Replicating heterochromatin regions were assigned to maternal or paternal domains on maximum-intensity projections by their enrichment in H3K9me3 and Ring1B, respectively.
See also Figures S1 and S2.
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4. Figure 2
PHC Reorganization Requires Major Satellite Transcription, but Not Progression through Replication
(A) Zygotes were collected and divided into two groups. The first group was injected between 24 and 27 hr phCG with LNA-DNA gapmers directed against GFP or a combination of forward and reverse major transcripts. The second group was placed in medium supplemented with 2.5 μg/ml aphidicolin or DMSO at 30 hr phCG. Both groups of embryos were cultivated in medium supplemented with 2 μM EdU from 30 hr phCG. Embryos were collected at ∼72 hr phCG and EdU incorporation was detected together with DNA FISH. At this time point, control embryos already cleaved to four cells.
(B) Control, aphidicolin-treated, and microinjected embryos were collected at ∼72 hr phCG and processed for EdU revelation and DNA FISH. Panels with respective enlargements show EdU staining (gray), as well as the predominant patterns of DNA FISH signals for minor (green) and major (red) satellites. Arrowhead indicates ring-like major satellites; arrow indicates major satellite DNA reorganized in chromocenters. The proportion of nuclei containing ring-like major satellites is displayed on the enlarged panels (p < 0.01 for aphidicolin versus LNA Maj1/2, Student’s t test). DNA was counterstained with DAPI (blue). Scale bar represents 10 μm.
(C) Aphidicolin-treated, microinjected embryos under aphidicolin treatment and α-amanitin/aphidicolin-treated embryos were collected at ∼72 hr phCG and processed for EdU revelation and DNA FISH. Panels with respective enlargements show EdU staining (gray), as well as DNA FISH for minor (green) and major (red) satellites. The proportion of nuclei containing ring-like major satellites is displayed on the enlarged panels. Binomial tests were applied to assess the significance of the differences observed between the treatments (all treatments compared with aphidicolin: not significant (NS) for LNA-GFP; p < 1.0 × 10−11 for LNA Maj1/2; p < 1.0 × 10−06 for α-amanitin). DNA was counterstained with DAPI (blue). Scale bar represents 10 μm.
See also Figures S2 and S3.
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5. Figure 3
Knockdown of Major Satellite Transcripts Impacts the Reorganization of Paternal and Maternal Heterochromatin in Two-Cell Embryos Differently
(A) Zygotes were injected between 24 and 27 hr phCG with LNA-DNA gapmers targeting either GFP or forward and reverse major satellite transcripts. Embryos were collected at ∼69 hr phCG and processed for immuno-DNA FISH.
(B) Microinjected embryos were stained for Ring1B (green, specifying the paternal heterochromatin) and processed for DNA FISH revealing major satellites (red).
(C) Similarly to (B), embryos were stained for H3K9me3 (green) to reveal the maternal PHC. DNA was counterstained with DAPI (gray). Scale bar represents 5 μm.
(D) Within the population of embryos with ring-like PHC domains, we assessed the percentage of two-cell nuclei in which the rings were of either paternal or maternal origin, based on the H3K9me3/Ring1B parental asymmetric staining. Error bars represent the SD from four independent experiments; ∗p < 0.05, ∗∗p < 0.01, Student’s t test.
(E) Two-cell embryos were collected at 45 and 50 hr phCG and stained for H3K9me3 (green, specifying the maternal genome) and processed for DNA FISH revealing major satellites (red). Arrowheads point to ring-like structures of major satellites. The percentage of nuclei containing ring-like major satellites enriched (maternal, ♀) or devoid (paternal, ♂) of H3K9me3 is indicated on the enlarged panels. Binomial tests were applied to test the significant increase in the presence of exclusively paternal ring structures between the two time points (paternal versus maternal: NS for 45 hr phCG, p < 1.0 × 10−12 for 50 hr phCG). DNA was counterstained with DAPI (gray). Scale bar represents 10 μm.
See also Figure S4.
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6. Figure 4
Parthenotes Require Major Satellite Transcripts for Developmental Progression and Proper Heterochromatin Consolidation
(A) Oocytes were collected at 16 hr phCG and activated for ∼6 hr in SrCl2 in the presence of cytochalasin B to generate diploid parthenotes. Parthenotes were injected with LNA gapmers targeting either GFP or forward and reverse major satellite transcripts and processed for DNA FISH at 42 hr postactivation (pa).
(B) Table representing the developmental phenotype of parthenotes microinjected with LNA-DNA gapmers.
(C) Noninjected and microinjected parthenotes were processed for DNA FISH with probes revealing major satellites (red). DNA was counterstained with DAPI (gray). Scale bar represents 5 μm. The percentage of parthenotes with ring-like major satellites after knockdown of major satellite transcripts is indicated.
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7. Figure 5
Knockdown of Reverse Major Satellite Transcripts Is Sufficient to Prevent Developmental Progression Past the Two-Cell Stage
(A) Zygotes were injected between 24 and 27 hr phCG with LNA-DNA gapmers directed against GFP or targeting either forward or reverse major satellite transcripts. Embryos were collected for RNA FISH at 42 hr phCG, for RNA extraction at ∼44–46 hr phCG, and for immuno-DNA FISH at ∼69 hr phCG.
(B) Quantification of the total intensity of the RNA FISH signals per nucleus for forward and reverse major satellite transcripts in two-cell embryos microinjected with LNA-DNA gapmers.
(C) qRT-PCR of major satellite transcripts after strand-specific reverse transcription. Mean of the expression levels of transcripts ± SD in two-cell embryos injected with LNA-DNA gapmers targeting either the forward or reverse major satellite RNAs compared with LNA-DNA gapmers targeting GFP and noninjected embryos from three independent experiments.
(D) Table representing the developmental phenotype of embryos microinjected with strand-specific LNA-DNA gapmers.
(E) Control and microinjected embryos were collected at ∼69 hr phCG and processed for DNA FISH. Panels with respective enlargements show DNA FISH for major satellites (red) of representative embryos for each set of LNA-DNA gapmers injected. DNA was counterstained with DAPI (gray). Close-ups illustrate major satellite organization in different groups of embryos. Scale bar represent 5 μm. The percentage of embryos with ring-like major satellites after knockdown of reverse major satellite RNA is indicated.
See also Figure S5.
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8. Figure 6
Parthenotes Require Reverse Major Satellite Transcripts for Proper Developmental Progression and Heterochromatin Organization at the Two-Cell Stage
(A) Oocytes were collected at 16 hr phCG and activated for ∼6 hr in SrCl2 in the presence of cytochalasin B to generate diploid parthenotes. After activation, parthenotes were injected with strand-specific LNA-DNA gapmers targeting either GFP or forward or reverse major satellite transcripts. At 42 hr pa, the parthenogenetic embryos were processed for DNA FISH.
(B) Table representing the developmental phenotype of parthenotes microinjected with strand-specific LNA-DNA gapmers.
(C) Parthenotes microinjected with LNA-DNA gapmers targeting GFP or forward or reverse major satellite transcripts were processed for DNA FISH with probes targeting major satellites (red). DNA was counterstained with DAPI (gray). Scale bar represents 10 μm. The percentage of parthenotes with ring-like major satellites after the strand-specific knockdown of major satellite transcripts is indicated.
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9. Figure S1
Replication Timing of Distinct Noncoding Regions in Two-Cell Embryos, Related to Figure 1
(A) Percentage of embryos showing early, mid and late S-phase patterns in the respective time frames during the 2-cell stage. EdU incorporation, together with DNA FISH for major satellite DNA, allowed us to distinguish three types of S-phase patterns (see Figures S1C and S1D). The first pattern is characterized by an EdU signal throughout the nucleus, with no detectable enrichment in PHC comparable to the early S-phase pattern in somatic cells. The second pattern shows the EdU signal in both euchromatin and PHC (termed mid S-phase pattern), while in the third pattern EdU mostly labels major satellites (late S-phase pattern).
(B) Ratio of the EdU signal in major satellites over the rest of the nuclei in 2-cell embryos at different stages of S-phase as quantified with the 3D-FIED macro. Values < 1 – no enrichment; values = 1 – similar levels; values > 1 enrichment.
(C) Embryos were pulsed for 1 hr with 50 μM EdU every hour from 31 hr to 43 hr phCG. At different time points during S-phase of the 2-cell stage (30-41 hr phCG), embryos were processed to reveal EdU incorporation (green), followed by DNA FISH for major satellites (red). DNA is counterstained with DAPI (gray). Scale bar represents 5 μm.
(D) Embryos were pulsed for 1 hr with 50 μM EdU every hour from 31 to 43 hr phCG. Immediately after each pulse, embryos were collected and processed for EdU detection (blue) together with DNA FISH for major satellites (red) and telomeres (green). DNA is counterstained with DAPI (gray). Scale bar represents 5 μm. Note how EdU is not colocalizing with telomeres during late S-phase, suggesting that these regions either start replication earlier than major satellites or that replication is resolved faster.
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10. Figure S2
Forward and Reverse Major Satellite RNAs Show Specific Transcriptional Dynamics during Preimplantation Development, Related to Figures 1 and 2
(A) Single plane representations of Figure 1B.
(B) RNA FISH for forward (green) and reverse (red) major satellite transcripts during the initial stages of pre-implantation development. Embryos were staged based on the timing of fixation (post-hCG) and morphology of the nucleus, NPBs and associated heterochromatin. Enlarged pictures show the paternal pronucleus of the zygote and representative nuclei for the posterior developmental stages. Before the first cell division, we detect some transcripts of both strands of major satellites, with a predominance of reverse transcripts, mostly in the male pronucleus. Scheme represents the expression patterns of major satellites and their replication (E = early, M = middle, L = late S-phase) dynamics from the zygote up to the second embryonic cleavage (4-cell stage). Note the peak of expression of the forward strand, predominantly from the paternal genome, followed by the peak of the reverse strand. Replication of paternal precedes maternal PHC domains.
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11. Figure S3
Blocking Two-Cell Embryos at G1/S with Aphidicolin Does Not Prevent Expression of Forward and Reverse Major Satellite Transcripts, Related to Figure 2
(A) Zygotes were cultured in the presence of 2.5 μg/ml aphidicolin from the end of the 1-cell stage. Embryos that cleaved to 2-cells were collected at around 42 hr phCG for RNA FISH. Panels with respective enlargements show RNA FISH for major forward (green) and major reverse (red) RNA in representative embryos belonging to control (DMSO) and aphidicolin treated groups. DNA was counterstained with DAPI (gray). Scale bar represents 10 μm.
(B) Quantitative RT-PCR of major satellite transcripts after aphidicolin treatment. Mean of the relative expression levels of transcripts ± SD in 2-cell embryos treated with aphidicolin compared to embryos treated with DMSO (control) from 2 independent experiments. Aphidicolin treatment does not prevent major satellite transcription, but seems to slightly affect the expression burst of the forward major satellite RNA, without impacting the reverse strand expression.
(C) Fluorescence images of embryos from the late zygote to late 2-cell stage, for which major satellites (red) have been revealed by DNA FISH. DNA is counterstained with DAPI (gray). Scale bar represents 5 μm. Image depicts the reorganization dynamics of PHC occurring during the two first stages of pre-implantation development.
(D) Zygotes were injected between 24-27 hr phCG with LNA-DNA gapmers targeting either GFP or forward and reverse major satellite transcripts. Embryos were collected for DNA FISH at 72 hr and 96 hr phCG. Panels with respective enlargements show DNA FISH for major satellites (red) and minor satellites (green) in representative embryos injected with each set of LNA-DNA gapmers. The proportion of nuclei showing ring-like major satellites is indicated on the enlarged panels, revealing the irreversible phenotype of microinjected embryos. DNA was counterstained with DAPI (gray). Scale bar represents 10 μm.
(E) Zygotes were cultured in the presence of 10 μg/ml α-amanitin from the end of the 1-cell stage. Embryos that cleaved to 2-cells were collected at around 42 hr phCG for RNA FISH. Panels with respective enlargements show RNA FISH for forward (green) and reverse (red) major satellite RNAs in representative embryos belonging to control and α-amanitin treated groups. The results show that the treatment with α-amanitin, efficiently prevents the expression of major satellite transcripts during the 2-cell stage. DNA was counterstained with DAPI (gray). Scale bar represents 10 μm.
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12. Figure S4
Major Satellite Transcripts Are Important for the Nuclear Rearrangement of Pericentric Heterochromatin during Preimplantation Development, Related to Figure 3
(A) Zygotes were injected between 24-27 hr phCG with LNA-DNA gapmers targeting either GFP or forward and reverse major satellite transcripts. Between 30-33 hr phCG, embryos were again injected with 2 ng/μl HP1α-HA mRNA. Embryos were collected for immunofluorescence at 46 hr phCG. Panels with respective enlargements show HA (red) and H3K9me3 (green) staining in representative embryos. DNA was counterstained with DAPI (gray). Scale bar represents 10 μm. Notice how recruitment of exogenous HP1α-HA is not affected when embryos are microinjected with gapmers targeting major satellite transcripts.
(B) Table representing the chromatin asymmetry (Ring1B/H3K9me3) of embryos injected with LNA-DNA gapmers.
(C) Single plane representations of Figures 3B and 3C.
(D) 2-cell embryos were collected between 45-50 hr phCG and stained for H3K9me3 (green, specifying the maternal genome) and processed for DNA FISH revealing major satellites (red). DNA was counterstained with DAPI (gray). Scale bar represents 10 μm. Single plane representations of the types of nuclear organization represented in Figure 3E. At 45 hr phCG, a similar percentage of nuclei show major satellite rings either devoid or enriched in H3K9me3. However, in late 2-cell embryos (50 hr phCG), in which most of major satellites are already organized in chromocenters, the remaining ring-like PHC is devoid of H3K9me3 in a significantly higher frequency than associated with this mark. This suggests that the reorganization of paternal PHC takes longer.
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13. Figure S5
LNA-DNA Gapmers Designed to Target Either Forward or Reverse Transcripts Efficiently Knock Down their Respective Targets, Related to Figure 5
(A) Zygotes were injected between 24-27 hr phCG with LNA-DNA gapmers targeting either GFP, forward or reverse major satellite transcripts. Embryos were collected for RNA FISH at around 42 hr phCG. Panels with respective enlargements show RNA FISH for forward (green) and reverse (red) major satellite RNA in representative embryos injected with each set of LNA-DNA gapmers. DNA was counterstained with DAPI (gray). Scale bar represents 5 μm.
(B) Quantitative Real-Time PCR of major satellite transcripts after strand-specific reverse transcription. Mean relative expression ± SD (from 3 independent experiments) in 2-cell embryos injected with different combinations and concentrations of LNA-DNA gapmers targeting either the forward, reverse or both major satellite RNAs normalized to LNA-DNA gapmers targeting GFP.
(C) Table representing the developmental phenotype of embryos microinjected with different concentrations of LNA-DNA gapmers.
Cell Reports 2013 4, DOI: ( /j.celrep )
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