HIF1A-mediated pathways promote euploid cell survival in chromosomally mosaic embryos
- Division of Biology 139-74, California Institute of Technology, United States
Figures
Figure 1 with 2 supplements
Lineage analysis of aneuploid embryos generated by selective Mps1 inhibitors AZ3146 and reversine.
(A) Graphic representation of 4-cell embryos treated with DMSO (control) or Mps1 inhibitors reversine (0.5 µM) and AZ3146 (20 µM) to inactivate the spindle assembly checkpoint (SAC) and induce chromosome segregation errors. After washing, embryos were cultured to the mature blastocyst stage (E4.5) and analyzed for lineage specification. (B) Immunofluorescence imaging of well-known lineage markers CDX2 (trophectoderm [TE]), NANOG (epiblast [EPI]), and SOX17 (primitive endoderm [PE]) reveals that overall embryonic morphology and cavitation is not affected by Mps1 inhibition. (C) Number of cells in each lineage was quantified to evaluate the effect of drug treatment on blastocyst development. Importantly, both reversine and AZ3146 treatments reduce the number of cells in the inner cell mass (ICM), marked by NANOG (EPI) and SOX17 (PE). Whereas the TE, marked by CDX2, is reduced only in the reversine-treated embryos (n=28 embryos per treatment, cumulative of three independent experiments. Statistical test: Mann–Whitney U-test, error bars represent s.e.m.). (D) Analysis of DNA damage and DNA repair based on immunofluorescence against γH2A.X (phosphorylated form of H2A.X, white) and PARP1 (red), respectively. (E) Intensity analysis shows that reversine and AZ3146 increase DNA damage at the 8-cell stage through morula stage compared with DMSO-treated embryos. Importantly, reversine appears to downregulate PARP1 expression at the 8-cell stage, which extends to morula stage embryos (n=~15 embryos per treatment, ~120 cells. Collection was made during three independent experiments. Statistical test: t-test, error bars represent s.e.m.).
Figure 1—figure supplement 1
AZ3146-treated embryos are aneuploid and can develop to post-implantation stages.
(A) In situ chromosome counting of 8-cell stage embryos after drug treatments. Representative images of individual embryos in the different treatments. DNA (white) is visualized by DAPI staining and kinetochores (red) by CREST antibody. PB (polar body), MN (micronuclei). Each chromosome presents two CREST dots. The large number is the cell ID, and the smaller number indicates the number of chromosomes, ‘n’. Diploid blastomeres present 40 pairs of CREST dots. Phalloidin (cyan) was used for cell segmentation. (B) Quantification of the incidence of aneuploid events, designated as those with more than two chromosomes (MN >2 chr), less than two chromosomes (MN <2 chr), without kinetochores (MN-CREST), and non-dividing nucleus (ND cell). We observed an increase in MN following reversine and AZ3146 treatment. Importantly, reversine-treated cells have a higher number of ND cells and MN-CREST staining (n=256-cell DMSO treatment, n=304 cells AZ3146 treatment, n=144 cells reversine treatment, n=~20 embryos per treatment. Statistical test: Mann–Whitney U-test, error bars represent s.e.m.). (C) Embryo transfer experiments show higher implantation events (% decidua formation) in AZ3146-treated embryos. The left uterine horn was used for transfer of reversine-treated blastocyst and the right uterine horn for AZ3146-treated blastocyst (n=7 embryos transplanted per uterine horn, repeated experiments two times). (D) Only AZ3146-treated blastocyst can develop post-gastrulation E9.5 fetuses, image representing three out of five surviving embryos (% embryo formation).
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Figure 1—figure supplement 1—source data 1
Aneuploidy quantification in embryos after in situ chromosome counting.
- https://cdn.elifesciences.org/articles/101912/elife-101912-fig1-figsupp1-data1-v1.xlsx
Figure 1—figure supplement 2
PARP1 is required for inner cell mass (ICM) development.
(A) Immunofluorescence against PARP1 (red) and γH2A.X (white) in blastocyst after drug treatments. Notice accumulation of DNA damage in reversine-treated blastocyst (representative images of n>12 embryos per treatment collected from three independent experiments). (B) Immunofluorescence against PARP1 (red), NANOG (white, epiblast [EPI]), and CDX2 (green, trophectoderm) at the blastocyst stage in untreated embryos. Analysis of PARP1 intensity shows an increase in the EPI lineage (n>21 embryos per treatment collected from three independent experiments. Statistical test: Mann–Whitney U-test, error bars represent s.e.m.). To confirm these observations, scRNA-seq (Deng et al., 2014) shows enrichment of Parp1 mRNA specifically in the EPI. (C) Graphic representation of 4-cell embryos treated with DMSO and aneuploid drugs. Downregulation of PARP1 was achieved by treatment with olaparib from morula to blastocyst stage. (D) Effects of the inhibition of PARP1 function were assessed by immunofluorescence against CDX2 (TE), NANOG (EPI), and SOX17 (primitive endoderm [PE]) in the different treatments. Importantly, olaparib treatment does not affect blastocyst morphology. (E) Lineage analysis at blastocyst stage shows a specific effect on the survival of EPI and PE cells in reversine-treated embryos. The ratio of cells in each lineage in the blastocyst is normalized based on DMSO treatment (n=15 embryos per treatment collected from three independent experiments. Statistical test: Mann–Whitney U-test, error bars represent s.e.m.).
Figure 2 with 1 supplement
AZ3146-treated embryos elevate HIF1A activity to support formation of the trophectoderm (TE) and primitive endoderm (PE).
(A) qPCR analysis of Trp53 and Hif1a mRNA expression at morula and blastocyst stages reveals that Trp53 is upregulated in reversine-treated embryos and that Hif1a is upregulated in AZ3146-treated embryos at morula stages (sample: three biological replicates and two technical replicates per experiment with each replicate having a minimum of 16 embryos. Statistical test: Welch’s t-test). (B) Immunofluorescence against HIF1A (black) shows an increase in nuclear intensity in AZ3146-treated embryos at morula stages. At the blastocyst stage, nuclear and cytoplasmic HIF1A are increased in AZ3146-treated embryos; normalization was based on DAPI staining (magenta) (n=~20 embryos per treatment. Statistical test: Mann–Whitney U-test, error bars represent s.e.m.). (C) Graphic representation of 4-cell embryos treated with DMSO and aneuploid drugs. Downregulation of HIF1A was achieved by treatment with IDF-11774 immediately after wash of the aneuploid drugs. Immunofluorescence analysis of lineage specification in blastocyst cultured with the HIF1A inhibitor IDF-11774 using antibodies against CDX2 (TE), NANOG (EPI), and SOX17 (PE). Importantly, IDF-11774 appears to affect cavitation of some AZ3146-treated embryos. (D) Lineage analysis at the blastocyst stage shows that TE and PE specification are affected by IDF-11774 treatment. The number of cells in each lineage was quantified to evaluate the effect on blastocyst development (n=20 embryos per treatment collected from three independent experiments. Statistical test: Mann–Whitney U-test, error bars represent s.e.m.).
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Figure 2—source data 1
qPCR quantifications for Trp53 and Hif1a in morulas and blastocysts.
- https://cdn.elifesciences.org/articles/101912/elife-101912-fig2-data1-v1.xls
Figure 2—figure supplement 1
Pharmacological inhibitors of HIF1A have distinct effects on mouse pre-implantation embryos.
(A) Inhibition of HIF1A from zygotes to blastocyst stage with PX-478 or IDF-11774 in normoxia. Immunofluorescence against CDX2 (trophectoderm [TE]) and DAPI (magenta) was used to assess blastocyst development. Importantly, IDF-11774 does not compromise pre-implantation development, whereas PX-478 compromised the survival and overall morphology of the blastocyst. (B) Analysis of the number of trophectoderm cells (CDX2) and total number (DAPI) shows a deleterious effect of PX-478 during blastocyst development (n=~12 embryos per treatment collected from three independent experiments. Statistical test: Mann–Whitney U-test, error bars represent s.e.m.).
Figure 3
Hypoxia exposure reduces DNA damage and affects lineage proportions in the aneuploid blastocyst.
(A) Graphic representation of the hypoxia experiments. 2-cell embryos were cultured until the blastocyst stage in hypoxia conditions (5% oxygen). As before, 4-cell stage embryos were treated with DMSO or Mps1 inhibitors reversine and AZ3146 until the 8-cell stage. After washing, embryos were cultured to the mature blastocyst stage (E4.5) and analyzed for lineage specification. (B) Immunofluorescence imaging of well-known lineage markers CDX2 (trophectoderm [TE]), NANOG (epiblast [EPI]), and SOX17 (primitive endoderm [PE]) reveals that overall embryonic morphology and cavitation is not affected by Mps1 inhibition or hypoxia. (C) Lineage analysis at blastocyst stage. The number of cells in each lineage was used to evaluate the effects in blastocyst development (n=18 embryos per treatment collected from three independent experiments. Statistical test: Mann–Whitney U-test, error bars represent s.e.m.). (D) Immunofluorescence against PARP1 (red) and γH2A.X (white) in blastocyst after drug treatments. (E) Intensity analysis shows that, under hypoxia, at 8-cell stage, DNA damage is increased after exposure to reversine but not AZ3146. PARP1 expression is altered only at the 8-cell stage in reversine-treated embryos (n=25 embryos per treatment collected from three independent experiments. Statistical test: t-test, error bars represent s.e.m.).
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Figure 3—source data 1
Raw fluorescence intensity of PARP1 and DAPI for reversine-treated embryos grown in normoxia.
- https://cdn.elifesciences.org/articles/101912/elife-101912-fig3-data1-v1.xls
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Figure 3—source data 2
Raw fluorescence intensity of PARP1 and DAPI for control embryos grown in normoxia.
- https://cdn.elifesciences.org/articles/101912/elife-101912-fig3-data2-v1.xls
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Figure 3—source data 3
Raw fluorescence intensity of PARP1 and DAPI for AZ3146-treated embryos grown in normoxia.
- https://cdn.elifesciences.org/articles/101912/elife-101912-fig3-data3-v1.xls
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Figure 3—source data 4
Raw fluorescence intensity of PARP1 and DAPI for reversine-treated embryos grown in hypoxia.
- https://cdn.elifesciences.org/articles/101912/elife-101912-fig3-data4-v1.xls
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Figure 3—source data 5
Raw fluorescence intensity of PARP1 and DAPI for control embryos grown in hypoxia.
- https://cdn.elifesciences.org/articles/101912/elife-101912-fig3-data5-v1.xls
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Figure 3—source data 6
Raw fluorescence intensity of PARP1 and DAPI for AZ3146-treated embryos grown in hypoxia.
- https://cdn.elifesciences.org/articles/101912/elife-101912-fig3-data6-v1.xls
Figure 4 with 2 supplements
Hypoxia affects cell competition between diploid and aneuploid cells during pre-implantation development.
(A) Graphic representation of the cell competition experiments. Embryos were treated at the 4-cell stage with DMSO or Mps1 inhibitors reversine and AZ3146. After washing, 8-cell stage embryos were disaggregated and re-aggregated to form chimeras containing a 1:1 ratio of DMSO/AZ314-treated blastomeres and reversine/AZ314-treated blastomeres. Following aggregation, chimeras were cultured to the mature blastocyst stage (E4.5) and analyzed for lineage specification. For identification of the treatment, we use transgenic lines with membrane markers. Immunofluorescence for CDX2, NANOG, and SOX17 was performed to test lineage specification and allocation during (B) normoxia and (C) hypoxia. Lineage allocation quantification was based on the above markers. Importantly, (D) in normoxia, both chimeras have the same number of cells in all the lineages except the epiblast (EPI). In addition, AZ3146-treated blastomeres outcompete reversine-treated blastomeres in medium-grade mosaics in the trophectoderm (TE) and the EPI. (E) Under hypoxia, both chimeras have a similar number of cells in all the lineages. Yet, AZ3146-blastomeres do not outcompete reversine-treated blastomeres. Quantification of the contribution of AZ3146-treated blastomeres to the chimeras showed that, (F) under normoxia, compared with DMSO-treated cells, no preferential allocation of aneuploid cells occurs in the TE. In contrast, AZ3146-treated blastomeres increased their contribution when compared with reversine-treated blastomeres, but only for the EPI and the primitive endoderm (PE). (G) During hypoxia, in DMSO/AZ3146 chimeras, no preferential allocation of aneuploid cells occurs in the TE. But preferential allocation of diploid cells to the EPI is observed. Whereas in reversine/AZ3146 chimeras, AZ3146-treated blastomeres contribute similarly to reversine blastomeres to the TE and PE but significantly increase contribution to the EPI. These results indicate that hypoxia favors the survival of reversine-induced aneuploid cells compared to their survival in normoxia (n>7 embryos per treatment collected from three independent experiments. Statistical test: Mann–Whitney U-test, error bars represent s.e.m.).
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Figure 4—source data 1
Quantification of lineage allocation in chimeric embryos.
- https://cdn.elifesciences.org/articles/101912/elife-101912-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Correlation plot of AZ3146-treated cells in the epiblast and trophectoderm.
Scatter plot of AZ3146-treated cells in epiblast and trophectoderm in DMSO/AZ3146 and reversine/AZ3146 mosaic blastocysts cultured under normoxic and hypoxic conditions.
Figure 4—figure supplement 2
Aneuploidies generated before the 2-cell stage do not affect lineage specification in mosaic embryos.
(A) Immunofluorescence for CDX2, NANOG, and SOX17 was performed in blastocysts after zygote treatment with Mps1 inhibitors. Importantly, reversine treatment at zygote stages affects morphology of blastocyst. (B) Lineage analysis of blastocyst shows no effect in AZ3146-treated embryos. Whereas reversine treatment reduces the cell number to almost half. The ratio of cells in each lineage in the blastocyst is normalized based on DMSO treatments (n=~27 embryos per treatment collected from three independent experiments. Statistical test: Mann–Whitney U-test, error bars represent s.e.m.). (C) Aggregation chimeras at 2-cell stage were created by using transgenic lines with membrane markers: mTmG (green) and E-cadherin (white). Subsequently, (D) immunofluorescence for CDX2, NANOG, and SOX17 was performed to quantify lineage allocation. Our results show no effect on embryo morphology in our chimeras. However, reversine/DMSO (D/R) chimeras seem to have increased events of cell extrusion, and reversine/AZ3146 chimeras are smaller in number of cells compared with DMSO/AZ3146 chimeras. (E) Data show the proportion of AZ3146-treated cells in DMSO/AZ3146 mosaic blastocysts (gray) and in reversine/AZ3146 mosaic blastocysts (blue), as well as the proportion of reversine-treated cells in DMSO/reversine mosaic blastocysts (red).
Figure 5 with 1 supplement
HIF1A inhibition increases the proportion of euploid cells in mosaic embryos.
(A) Schematic of HIF1A inhibition by IDF-11774 in 8-cell stage aggregation chimeras cultured in hypoxia. Immunofluorescence for CDX2, NANOG, and SOX17 was performed to test lineage specification and allocation. (B) HIF1A inhibition in low-grade mosaicism does not affect overall morphology but affects (C) cavity diameter. In addition, (D) lineage allocation quantification reveals a significant reduction of total cell number, as well as a reduction in cell number in the TE but not in the epiblast (EPI) and primitive endoderm (PE). In contrast, (E) HIF1A inhibition in medium-grade mosaicism affects morphology and (F) cavitation of the mosaic embryos. However, lineage allocation quantification revealed that total cell number is not affected (G) in medium-grade mosaicism after IDF11-774 treatment. Nevertheless, HIF1A inhibition appears to increase the cell number of the PE. These results show that HIF1A inhibition in hypoxic conditions differentially affects each type of mosaic embryos (n>8 embryos per treatment collected from three independent experiments. Statistical test: Mann–Whitney U-test, error bars represent s.e.m.).
Figure 5—figure supplement 1
IDF-11774-mediated inhibition of HIF1A increases the proportion of euploid cells in mosaic embryos.
Bright-field images of low- and medium-mosaic chimeras grown in normoxia and treated with (A) DMSO and (B) IDF-11774. Quantification of the contribution of AZ3146-treated blastomeres to the chimeras showed that, (C) in DMSO/AZ3146 mosaics, IDF-11774 treatment reduces the proportion of AZ3146-treated blastomeres, whereas (D) in reversine/AZ3146 mosaics, IDF-11774 treatment slightly increases the proportion of AZ3146-treated blastomeres. These results may indicate that HIF1A inhibition impairs the less fit population in chimeric embryos.
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HIF1A-mediated pathways promote euploid cell survival in chromosomally mosaic embryos
eLife 13:RP101912.
https://doi.org/10.7554/eLife.101912.4
