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 (TE), NANOG (EPI) and SOX17 (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 ICM, marked by NANOG (EPI) and SOX17 (PE). Whereas the TE, marked by CDX2, is reduced only in the reversine-treated embryos. (n=28 per treatment) (***P<0.0001, **P<0.01, *P<0.05, 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. Importantly, reversine appears to downregulate PARP1 expression at the 8-cell stage, which extends to morula stage embryos (***P<0.0001, **P<0.01, *P<0.05, t-test).

AZ3146-treated embryos elevate HIF1A activity to support formation of the TE and PE.

A) qPCR analysis of p53 and Hif1a mRNA expression at morula and blastocyst stages reveals that p53 is upregulated in reversine-treated embryos and that Hif1a is upregulated in AZ3146-treated embryos at morula stages; 3 biological replicates and 2 technical replicates per experiment with each replicate having a minimum of 16 embryos (**P<0.01, *P<0.05, Welch’s t-test). B) Immunofluorescence against HIF1A (black) shows an increase in nuclear intensity in AZ3146-treated embryos at morula stages. At blastocyst stage, nuclear and cytoplasmic HIF1A are increased in AZ3146-treated embryos; normalization was based on DAPI staining (magenta). (C) Graphic representation of 4-cell embryos treated with DMSO and aneuploid drugs. Chemical 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. Number of cells in each lineage was quantified to evaluate the effect in blastocyst development. (***P<0.0001, **P<0.01, *P<0.05, Mann–Whitney U-test, error bars represent s.e.m).

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 (TE), NANOG (EPI) and SOX17 (PE) reveals that overall embryonic morphology and cavitation is not affected by Mps1 inhibition or hypoxia. (C) Lineage analysis at blastocyst stage. Number of cells in each lineage was used to evaluate the effects in blastocyst development. (*P<0.05, 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, DNA damage is only slightly increased at the 8-cell stage after exposure to reversine and AZ3146. PARP1 expression is altered only at the 8-cell stage in reversine-treated embryos (***P<0.0001, **P<0.01, *P<0.05, t-test).

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/DMSO-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 transgenics 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 EPI. In addition, AZ3146-treated blastomeres outcompete reversine-treated blastomeres in medium-grade mosaics in the TE and the EPI. E) Under hypoxia, both chimeras have 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, compare with DMSO-treated cells, no preferential allocation of aneuploid cells occurs in the TE. In contrast, AZ3146-treated blastomeres increased their contribution when compare with reversine-treated blastomeres, but only significantly on the EPI and the PE. (G) During hypoxia, DMSO/AZ3146 chimeras did not change their behavior. Confirming a preferential allocation of diploid cells to the EPI. In reversine/AZ3146 chimeras, AZ3146-treated blastomeres contribute similarly to reversine-blastomeres to the TE and EPI but significantly increase contribution to the EPI. These results indicate that hypoxia favors the survival of reversine-induced aneuploid cells (Experiments were repeated in triplicate, ****P<0.00001, **P<0.01, *P<0.05, Mann–Whitney U-test, error bars represent s.e.m).

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) cavitation. In contrast, (E) HIF1A inhibition in medium-grade mosaicism affects morphology, and (F) cavitation of the embryos. Lineage allocation quantification after IDF-1174 reveals a significant reduction of total cell number (D) in low-grade mosaicism as well as a reduction in cell number in the TE but not in the EPI and PE. In contrast, total cell number is not affected (G) in medium-grade mosaicism. However, IDF11-774 treatment appears to increase the cell number of the PE. These results show that HIF1A inhibition in hypoxic conditions differentially affect each type of mosaic embryos. Experiments were repeated in triplicate, **P<0.005, *P<0.05, Mann–Whitney U-test, error bars represent s.e.m.

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 2 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 2 chromosomes (MN>2chr), less than 2 chromosomes (MN<2chr), 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=128 cell DMSO treatment, n=152 cells AZ3146 treatment, n=72 cells reversine treatment, ****P<0.00001, **P<0.01, *P<0.05, 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. (D) Only AZ3146-treated blastocyst can develop post-gastrulation E9.5 fetuses (% embryo formation).

PARP1 is required for 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. (B) Immunofluorescence against PARP1 (red), NANOG (white, epiblast) and CDX2 (green, trophectoderm). Analysis of PARP1 intensity shows an increase in the EPI lineage. To confirm these observation, 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. Chemical 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 (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 epiblast and PE cells in reversine-treated embryos. Ratio of cells in each lineage in the blastocyst is normalized based on DMSO treatment. (***P<0.0001, **P<0.01, *P<0.05, Mann–Whitney U-test, error bars represent s.e.m).

Pharmacological inhibitors of HIF1A have distinct effects on mouse pre-implantation embryos.

(A) Chemical inhibition of HIF1A from zygotes to blastocyst stage with PX-478 or IDF-11774. Immunofluorescence against CDX2 (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 (***P<0.0001, **P<0.01, *P<0.05, Mann–Whitney U-test, error bars represent s.e.m).

Correlation plot of AZ3146-treated cells in the epiblast and trophectoderm.

Scatter plot of AZ3146-treated cells in epiblast and trophectoderm in DMSO/AZ3146 mosaic blastocysts.

Aneuploidies generated at different stages have similar consequences on post-implantation development in vitro.

(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. Ratio of cells in each lineage in the blastocyst is normalized based on DMSO treatments. (n>27 per treatment) (***P<0.0001, **P<0.01, *P<0.05, Mann–Whitney U-test, error bars represent s.e.m). (C) Aggregation chimeras at 2-cell stage were created by using transgenics 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 seems to have increase events of cell extrusion and reversine/AZ3146 chimeras are smaller in number of cells compare with DMSO/AZ3146 chimeras. (E) Data show the proportion of AZ3146-treated cells in DMSO/AZ3146 mosaic blastocysts (grey) and in reversine/AZ3146 mosaic blastocysts (blue), as well as the proportion of reversine-treated cells in DMSO/reversine mosaic blastocysts (red).

IDF-11774-mediated inhibition of HIF1A increases the proportion of euploid cells in mosaic embryos.

Brightfield images of low- and medium-grade chimeras grown in (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.