HIF1A contributes to the survival of aneuploid and mosaic pre-implantation embryos

Estefania Sanchez-Vasquez, Marianne E. Bronner, Magdalena Zernicka-Goetz

Posted on: 11 October 2024

Preprint posted on 19 July 2024

Hypoxia regulates embryonic fitness in mosaic aneuploid embryos

Selected by Anchel De Jaime Soguero

Categories: cell biology, developmental biology, genetics, molecular biology

Background:

Human embryo development is an inefficient process in which two out of three embryos will not develop. The degree of chromosomal mosaicism (presence of euploid and aneuploid cells in the embryo) correlates with developmental fitness, implantation and survival of the embryo ^1–3. However, how the microenvironment affects mosaicism, and how the embryo manages it is currently unknown. Addressing these points becomes critical not only to understand the molecular processes underlying embryonic robustness, but also to improve human embryo culture and fitness for reproductive medicine purposes.

Given the ethical concerns of working with human preimplantation embryos, the authors of this preprint turned to the mouse embryo as a model in this study. Of note, chromosomal mosaicism drastically differs between human (70%) and mouse (25%) preimplantation embryos in normal conditions^3,4. To overcome this species-specific issue, the authors used two different MSP1 checkpoint inhibitors (Reversine and AZ3146) which trigger aneuploidy and mosaicism in the cleaving mouse embryos. Under this approach, the authors aimed to elucidate: i) what the fate is of aneuploid cells in the embryo (lineage wise) and ii) how hypoxia conditions affect developmental fitness of aneuploid and euploid cells.

Methods in a nutshell:

The authors cultured in vitro mouse preimplantation embryos. Between the 4-cell to 8-cell stage,chromosomal mosaicism was induced using MSP1 inhibitors. Afterwards, the medium was washed and the authors left the embryos to develop until late blastocyst stage (E4.5). Most of the experimental approach revolved around immunofluorescence staining of the embryos; the authors assessed, among different things, i) the presence of micronuclei and chromosome number imbalances by arresting cells in metaphase; ii) the DNA damage and repair activity in the embryo (by means of PARP1 and gH2AX expression, respectively); iii) the localization of HIF1a protein; as well as iv) the contribution of early embryonic cells towards the Epiblast, Primitive endoderm and Trophectoderm (staining for NANOG, SOX17 and CDX2 markers, respectively). In addition, the authors generated chimeras of euploid vs. mosaic cells from fluorescent-tagged mouse embryos to understand cellular competition across preimplantation development.

Key findings:

AZ3146 and Reversine MSP1 inhibitors trigger aneuploidy but also differential stress responses in the embryo.

The authors could confirm that MSP1 inhibition leads to aneuploidy and mosaicism associated events such as micronuclei formation in the embryo, as previously reported⁵. However, they observed an intriguing difference between both MSP1 inhibitors tested; while aneuploidy caused by reversine is accompanied by p53-dependent apoptosis and a sustained DNA damage response through morula and blastocyst stages, AZ3146 did not trigger a p53 response. In turn, AZ3146 increased HIF1a expression across preimplantation development. Furthermore, by performing challenging embryo transfer experiments, the authors could prove that reversine-treated embryos cannot develop, while 20% of AZ3146-treated embryos were born after transfer to the mother’s decidua. This suggests that differential stress responses of MSP1 inhibitors, rather than mosaicism or aneuploidy itself, controls embryo competence.

Hypoxia balances chromosomal mosaicism by cell competition between aneuploid and euploid embryonic cells.

Previous data suggest apoptotic clearance of Epiblast aneuploid cells in embryos treated with reversine⁵. By comparing both MSP1 inhibitor treatments, the authors sought to understand if this is something driven by aneuploidy or secondary effects of the drugs. Given the specific upregulation of the hypoxic master regulator HIF1a in embryos treated with AZ3146, the authors hypothesized that hypoxia might control aneuploid cell fate leading to embryo competence. After performing chimeric embryo experiments, they concluded that hypoxia supports epiblast euploid cell fitness over aneuploid cells, but not in extraembryonic lineages (trophoblast and primitive endoderm). Later on, they demonstrate that this effect is HIF1a specific. These results suggest that hypoxia enhances developmental competence by supporting euploid cellular fitness in the epiblast.

Why I chose this preprint?

I am very interested in understanding how cellular lineages respond to microenvironmental changes and sources of cellular stress in the context of development. The causes of the elevated rate of spontaneous human miscarriages per pregnancy are poorly understood, in great part due to the lack of human embryo availability for research purposes. This translates to enormous economic loss in the health care sector, as well as psychological and sociological hurdles that the pregnant mother and family have to overcome.

I like the approach taken by the authors to mimic mosaicism in the mouse embryo, and how they link a critical environmental factor like hypoxia to mosaicism, lineage specification and embryo viability. In fact, hypoxic conditions surround the embryo both in vivo (in the uterus) as well as in vitro during human in vitro fertilization (IVF) cycles, and it is perhaps not surprising how this can support the fittest conditions for embryo growth. I believe the next steps of this story should uncover the molecular mechanisms linking hypoxia and mosaicism in the embryo, and how this translates to physiological human embryo mosaic conditions.

Questions to the authors:

1) What are the aneuploidy rates of AZ3146 vs. Reversine treatment, beyond micronuclei formation? How many of the mitosis events under these treatments show imbalances in the number of chromosomes? I think this is an important quantification to understand the downstream effects of both MSP1 inhibitors.

2) It is very interesting to see how AZ3146 treatment does not seem to affect p53 signaling as reversine treatment does; however, relying only on TP53 mRNA levels can be misleading. Have you checked p53 Ser-15 phosphorylation or downstream targets of the cascade? Did you compare apoptotic rates between treatments? In a recent article, Regin et al. ⁶ demonstrate that aneuploidy-driven by Reversine triggers proteotoxic stress and autophagy; do you think similar events occur in AZ3146 treatment?

3) Recent discoveries point to the elevated replicative stress in the first cleavages as a source of chromosomal mosaicism in human embryos (Palmerola et al. Cell (2022)⁷). Besides this, MSP1 inhibition leads to massive and unscheduled aneuploidy. Do you think that triggering mild replicative stress (ie. with low doses of the DNA polymerase inhibitor Aphidicolin) could be a better option to mimic mosaicism than MSP1 inhibition?

4) Why do you think the epiblast does not tolerate the presence of aneuploid cells as well as extraembryonic lineages?

5) Do you think your findings translate well to human embryos? How comparable is physiological human embryo mosaicism to MSP1-I driven mosaicism in mouse embryos?

References:

Capalbo, A. & Rienzi, L. Mosaicism between trophectoderm and inner cell mass. Fertility and Sterility vol. 107 1098–1106 (2017).
Mccoy, R. C. Mosaicism in Preimplantation Human Embryos: When Chromosomal Abnormalities Are the Norm. (2017) doi:10.1016/j.tig.2017.04.001.
Vanneste, E. et al. Chromosome instability iscommon in human cleavage-stage embryos. Nat. Med. Vol. 15, (2009).
Lightfoot, D. A., Kouznetsova, A., Mahdy, E., Wilbertz, J. & Höög, C. The fate of mosaic aneuploid embryos during mouse development. Dev. Biol. 289, 384–394 (2006).
Bolton, H. et al. Mouse model of chromosome mosaicism reveals lineage-specific depletion of aneuploid cells and normal developmental potential. Nat. Commun. 2016 71 7, 1–12 (2016).
Regin, M. et al. Complex aneuploidy triggers autophagy and p53-mediated apoptosis and impairs the second lineage segregation in human preimplantation embryos. Elife 12, (2023).
Palmerola, K. L. et al. ll Replication stress impairs chromosome segregation and preimplantation development in human embryos. Cell 185, 2988–3007 (2022).

Tags: aneuploidy, cell fate, human embryo, hypoxia

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Author's response

The author team shared

1. What are the aneuploidy rates of AZ3146 vs. Reversine treatment, beyond micronuclei formation? How many of the mitosis events under these treatments show imbalances in the number of chromosomes? I think this is an important quantification to understand the downstream effects of both MSP1 inhibitors.

This is a good point we hope can be addressed in future. While there do seem to be other differences between the response to these two Mps1 inhibitors, this would need to be explored further to determine whether these are consequences of aneuploidy or off-target effects. Interestingly, one of these effects, an apparent increase in the DNA damage response actually closely recapitulates events observed in early development. We now know that replication stress is very common during the first cleavage divisions (7), and this may be the cause of the spontaneous DNA damage observed early in development. Thus, we aimed to understand the relationship between DNA damage, mosaic development, and survival of the pre-implantation embryos. Early embryos are regulative at all levels, and our evidence suggests that embryos may activate lineage-specific DNA repair mechanisms. This suggests the trophectoderm may be more resistant to aneuploid stress and knock on effects, in contrast to the epiblast which seems more sensitive. Thus, this is a very interesting finding.

2. It is very interesting to see how AZ3146 treatment does not seem to affect p53 signaling as reversine treatment does; however, relying only on TP53 mRNA levels can be misleading. Have you checked p53 Ser-15 phosphorylation or downstream targets of the cascade? Did you compare apoptotic rates between treatments? In a recent article, Regin et al. ⁶ demonstrate that aneuploidy-driven by Reversine triggers proteotoxic stress and autophagy; do you think similar events occur in AZ3146 treatment?

Thank you for raising this interesting point. We found that p53 is not the only protein that shows differential effects following MSP1 inhibitor treatments. We observed that AZ3146-treated embryos resolve DNA damage faster than those treated with Reversine. This difference may be related to the higher levels of PARP1 and HIF1a observed in AZ3146-treated embryos, which could account for the distinct dynamics in the DNA damage response.

We agree that it would be valuable to check whether AZ3146 treatment results in fewer apoptotic cells in the epiblast compartment, given that Reversine-treated embryos showed a reduced number of cells overall.

As to whether the unfolded protein response or autophagy is present in the epiblast of AZ3146-treated embryos, similar to what was reported for Reversine treatment, this would be an interesting area to explore in the future. However, it was not our priority for this study, as it does not address the differences in HIF1a levels and DNA damage response dynamics between the two treatments.

3. Recent discoveries point to the elevated replicative stress in the first cleavages as a source of chromosomal mosaicism in human embryos (Palmerola et al. Cell (2022)⁷). Besides this, MSP1 inhibition leads to massive and unscheduled aneuploidy. Do you think that triggering mild replicative stress (ie. with low doses of the DNA polymerase inhibitor Aphidicolin) could be a better option to mimic mosaicism than MSP1 inhibition?

The research from Dr. Egli’s group is remarkable for the field. For aphidicolin, and other approaches that increase replication stress, our concern was that there is evidence that transcription takes place coordinated with DNA replication. To avoid any extra effect on gene expression independent of an increase in DNA damage caused by inhibition of the SAC, we decided to create our models based on MPS1 inhibitors. Importantly, a major goal was to test cell competition in the chimeric mouse embryo.

It is important to note that aneuploidy typically arises from errors in chromosome segregation that occurs during cleavage division in the embryo, such as defects in the spindle assembly checkpoint. As a consequence those cells might activate the DNA damage or stress response. In contrast, triggering mild replicative stress with DNA polymerase inhibitors, can cause chromosome instability with structural changes and/or aneuploidies as a direct cause of the DNA damage and stress response. Therefore, in our opinion, MSP1 inhibitors are a more accurate way to replicate the natural embryo development. Finally, MSP1 inhibitors cause a relatively similar level of aneuploid cells in the mouse embryo compared with the human (please see also comments below.

4. Why do you think the epiblast does not tolerate the presence of aneuploid cells as well as extraembryonic lineages?

We believe this is a consequence of evolutionary paths that protect the pluripotency of the epiblast. Any mistake in the embryonic lineage will affect the embryo and progeny cells, so it is not surprising that mechanisms have evolved to avoid the propagation of aneuploidies/errors. In the context of the mechanisms, we observed an overexpression of the DNA damage sensor PARP1 in the epiblast, and this may be generating more sensitivity to DNA damage. Moreover, we have shown in our earlier work that the epiblast cells exhibit mechanisms of cell competition, which actively eliminate aneuploid cells to ensure the integrity and viability of the developing embryo.

In contrast, it remains unclear whether extraembryonic tissues, such as the trophoblast or primitive endoderm, exhibit cell competition to the same extent. The difference in tolerance to aneuploid cells may therefore be due to the epiblast’s crucial role in forming the entire foetus, with stricter quality control checkpoints to prevent abnormalities.

5. Do you think your findings translate well to human embryos? How comparable is physiological human embryo mosaicism to MSP1-I driven mosaicism in mouse embryos?

The data from Regin et al showed that, similar to our models, p53 and hypoxia pathways are activated in human aneuploid embryos. However, Capalbo et al 2021 already showed that low-to-medium mosaic aneuploid embryos can give rise to successful pregnancy at the same percentages as fully euploid embryos.

It is important to note that the percentage of human embryos with some degree of mosaicism can be quite high, ranging from 50% to 80%. On average, mosaic human embryos have aneuploid cells at a frequency of 20% to 50%. Similarly, treatment with Reversine in mouse embryos induces aneuploidy in about 50% to 60% of cells, which falls within the upper range of mosaicism reported in human embryos.

However, the specific types and frequencies of chromosome imbalances may differ between aneuploidies found in human embryos and those induced by MSP1 inhibitor treatment. This difference could impact direct comparisons between these models. That said, we believe that comparisons between our models are going to help understand the mechanisms that allow the survival of mosaic embryos.

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