Asynchronous mouse embryo polarization leads to heterogeneity in cell fate specification
Curation statements for this article:-
Curated by eLife
eLife assessment
This important work has substantially advanced our understanding of the molecular basis of symmetry breaking and lineage specification in preimplantation mammalian embryos. The results generated using live imaging are compelling. Quantification of the functional assays is convincing and would be improved by increasing the number of embryos in the evaluations and clearly stating how many embryos are evaluated per experiment.
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Abstract
The first lineage allocation in mouse and human embryos separates the inner cell mass (ICM) from the outer trophectoderm (TE). This symmetry breaking event is executed through polarization of cells at the 8-cell stage and subsequent asymmetric divisions, generating polar (TE) and apolar (ICM) cells. Here, we show that embryo polarization is unexpectedly asynchronous. Cells polarizing at the early and late 8-cell stage have distinct molecular and morphological properties that direct their following lineage specification, with early polarizing cells being biased towards producing the TE lineage. More recent studies have also implicated heterogeneities between cells prior to the 8-cell stage in the first lineage allocation: cells exhibiting reduced methyltransferase CARM1 activity at the 4-cell stage are predisposed towards the TE fate. Here, we demonstrate that reduced CARM1 activity and upregulation of its substrate BAF155 promote early polarization and TE specification. These findings provide a link between asymmetries at the 4-cell stage and polarization at the 8-cell stage, mechanisms of the first lineage allocation that had been considered separate.
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eLife assessment
This important work has substantially advanced our understanding of the molecular basis of symmetry breaking and lineage specification in preimplantation mammalian embryos. The results generated using live imaging are compelling. Quantification of the functional assays is convincing and would be improved by increasing the number of embryos in the evaluations and clearly stating how many embryos are evaluated per experiment.
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Reviewer #1 (Public review):
Summary:
This work starts with the observation that embryo polarization is asynchronous starting at the early 8-cell stage, with early polarizing cells being biased towards producing the trophectoderm (TE) lineage. They further found that reduced CARM1 activity and upregulation of its substrate BAF155 promote early polarization and TE specification, this piece of evidence connects the previous finding that at Carm1 heterogeneity 4-cell stage guide later cell lineages - the higher Carm1-expressing blastomeres are biased towards ICM lineage. Thus, This work provides a link between asymmetries at the 4-cell stage and polarization at the 8-cell stage, providing a cohesive explanation regarding the first lineage allocation in mouse embryos.
Strengths:
In addition to what has been put in the summary, the advanced …
Reviewer #1 (Public review):
Summary:
This work starts with the observation that embryo polarization is asynchronous starting at the early 8-cell stage, with early polarizing cells being biased towards producing the trophectoderm (TE) lineage. They further found that reduced CARM1 activity and upregulation of its substrate BAF155 promote early polarization and TE specification, this piece of evidence connects the previous finding that at Carm1 heterogeneity 4-cell stage guide later cell lineages - the higher Carm1-expressing blastomeres are biased towards ICM lineage. Thus, This work provides a link between asymmetries at the 4-cell stage and polarization at the 8-cell stage, providing a cohesive explanation regarding the first lineage allocation in mouse embryos.
Strengths:
In addition to what has been put in the summary, the advanced 3D image-based analysis has found that early polarization is associated with a change in cell geometry in blastomeres, regarding the ratio of the long axis to the short axis. This is considered a new observation that has not been identified.
Weaknesses:
For the microinjection-based method to overexpression/deletion of proteins, although it has been shown to be effective in the early embryo settings and has been widely used, it may not fully represent the in vivo situation in some cases, compared to other strategies such as the use of knock-in mice. This is a minor weakness; it would be good to include some sentences in the discussion on the potential caveats.
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Reviewer #2 (Public review):
Summary:
In this study, Lamba and colleagues suggest a molecular mechanism to explain cell heterogeneity in cell specification during pre-implantation development. They show that embryo polarization is asynchronous. They propose that reduced CARM1 activity and upregulation of its substrate BAF155 promote early polarization and trophectoderm specification.
Strengths:
The authors use appropriate and validated methodology to address their scientific questions. They also report excellent live imaging. Most of the data are accompanied by careful quantifications.
Weaknesses:
I think this manuscript requires some more quantification, increased number of embryos in their evaluations and clearly stating the number of embryos evaluated per experiments.
Here are some points:
(1) It should be clearly stated in all …
Reviewer #2 (Public review):
Summary:
In this study, Lamba and colleagues suggest a molecular mechanism to explain cell heterogeneity in cell specification during pre-implantation development. They show that embryo polarization is asynchronous. They propose that reduced CARM1 activity and upregulation of its substrate BAF155 promote early polarization and trophectoderm specification.
Strengths:
The authors use appropriate and validated methodology to address their scientific questions. They also report excellent live imaging. Most of the data are accompanied by careful quantifications.
Weaknesses:
I think this manuscript requires some more quantification, increased number of embryos in their evaluations and clearly stating the number of embryos evaluated per experiments.
Here are some points:
(1) It should be clearly stated in all figure legends and in the text how many cells from how many embryos were analyzed.
(2) I think that the number of embryos sometimes are too low. These are mouse embryos easily accessible and the methods used are well established in this lab, so the authors should make an effort to have at least 10/15 embryos per experiment. For example "In agreement with this, hybridization chain reaction (HCR) RNA fluorescence in situ hybridization of early 8-cell stage embryos revealed that the number of CDX2 mRNA puncta was higher in polarized blastomeres with a PARD6-positive apical domain than in unpolarized blastomeres, for 5 out of 6 embryos with EP cells (Figure 3A, B)".. or the data for Figure 4, we know how many cells but now how many embryos.
(3) It would be useful to see in Figure 4 an example of asymmetric cell division as done for symmetric cell division in panel 4B. This could really help the reader to understand how the authors assessed this.
(4) Figure 5C there is a big disproportion of the number of EP and LP identified. Could the authors increase the number of embryos quantified and see if they can increase EP numbers?
(5) Could the authors give more details about how they mount the embryos for live imaging? With agarose or another technique? In which dishes? Overlaid with how much medium and oil? This could help other labs that want to replicate the live imaging in their labs. Also, was it a z-stack analysis? If yes, how many um per stack? Ideally, if they also know the laser power used (at least a range) it would be extremely useful.
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