Totipotency: A developmental insurance policy

Why does a totipotent state linger within the inner cell mass of mouse embryos?.
  1. Nestor Saiz
  2. Anna-Katerina Hadjantonakis  Is a corresponding author
  1. Memorial Sloan Kettering Cancer Center, United States

The very first decisions in the life of a mammal are made even before the embryo implants into the womb. During this time, as the number of cells in the embryo increases from one to two to four and so on, the cells start to specialize to form distinct lineages. The first choice a cell faces is whether to join a cell population called the inner cell mass and become part of the embryo, or to join the trophectoderm lineage and become part of the placenta.

The biology of this cell fate decision has been a subject of intrigue and experimental pursuit for over half a century. Building on landmark work by the late Krystof Tarkowski, Martin Johnson and others (Johnson and Ziomek, 1981; Tarkowski and Wróblewska, 1967), recent studies have demonstrated the importance of the Hippo signaling pathway – a pathway well known for regulating cell growth and death – in this process (reviewed in Sasaki, 2017). These studies have established how the polarity and position of a cell either cause activation of the Hippo pathway in the inner cells of the embryo, or inhibit it in the outer cells of the embryo to promote the expression of genes encoding a trophectoderm identity.

Previous attempts to determine the exact timing of when cells commit to either the inner cell mass (ICM) or the trophectoderm (TE) lineage yielded somewhat conflicting results. Now, in eLife, Janet Rossant and colleagues – including Eszter Posfai of the Hospital for Sick Children in Toronto as first author – report how they have used the thread and needle of fluorescent reporters and single-cell transcriptomics to stitch together classic and recent findings on this topic (Posfai et al., 2017).

A transcription factor called CDX2 has a central role in triggering the TE transcriptional program. The expression of CDX2 in cells that go on to become part of the TE relies on a complex called TEAD-YAP, which is activated by inhibition of the Hippo pathway in the outer cells of the embryo (Nishioka et al., 2009). Posfai et al. used a CDX2-GFP fusion (McDole and Zheng, 2012) to sort CDX2-positive and CDX2-negative cells, followed by single-cell RNA sequencing, to determine how the TE and ICM transcriptional programs became established as the embryo developed from the 16-cell stage to the 32-cell stage.

These data raise the question of what the progressive stabilization of cell fate might tell us about commitment to either lineage. Could the expression of TE genes restrict cells to a TE fate even when challenged experimentally (i.e., when placed in a new context)? In the assays used to test these questions, either single cells have to be implanted into genetically-distinct host embryos to generate a chimera, or an embryo needs to be rebuilt from isolated cells of one particular type (inner or outer). The contribution of daughter cells to the resulting embryo will reveal details about lineage commitment in the parental cells. With these techniques, the labs of Tarkowski, Johnson and Rossant previously established that both the inner and outer cells remain totipotent – that is, they can give rise to the ICM and TE lineages – until the 16-cell stage, with some inner cells remaining totipotent until the 32-cell stage (Suwińska et al., 2008; Rossant and Vijh, 1980; Ziomek et al., 1982).

Posfai et al. perform a contemporary version of these classic experiments using the CDX2-GFP fluorescent genetic marker, rather than cell position, to discriminate between prospective TE and ICM cells. They were able to precisely match cell fate commitment to the relevant gene expression profile of individual cells for both (GFP-positive and GFP-negative) populations at successive stages of development. This allowed them to confirm previous results and to paint a detailed picture of the molecular players that are potentially involved in stabilizing these two cell fates. For cells expressing CDX2, cell fate is basically sealed soon after blastocyst formation (at the 32-cell stage), and they are unable to give rise to the ICM. ICM cells, on the other hand, delay their commitment by an additional cell cycle, up until some point between the 32- and 64-cell stages (Figure 1). Posfai et al. confirm these results by genetically and pharmacologically modulating the activity of the Hippo pathway, which connects apico-basal cell polarity (or the lack of it in ICM cells) to gene expression.

Cell differentiation in mammalian embryos.

Cells that develop into the trophectoderm (TE) express the transcription factor CDX2 (denoted here as CDX2+) and commit to their cell fate at around the 32-cell stage. Cells that will develop into the inner cell mass (ICM) keep their options open and only commit to their cell fate at a time between the 32- and 64-cell stage; these cells do not express CDX2 (denoted here as CDX2-). As embryos grow from the 32- to the 64-cell stage, ICM cells start to differentiate into two new cell lineages: the embryonic epiblast (future fetus) and the extra-embryonic primitive endoderm (future yolk sac).

This raises the question of why commitment to the ICM lineage takes place later during development. Perhaps it is no coincidence that ICM cells gradually begin to make their next cell fate choice during this time window. It is at this time that ICM cells make a decision to become epiblast (future fetus) versus primitive endoderm (future yolk sac). Therefore, the ICM may not be a cell fate per se, but rather a transitory state that lasts only until all the cells in the embryo have been allocated to one of the three lineages that make up the blastocyst. An asynchrony in making these early fate decisions could therefore reflect a developmental insurance policy: a strategy to guarantee that enough cells differentiate for each of the cell types that lay the foundation for all embryonic and extra-embryonic tissues (Saiz et al., 2016).


    1. Tarkowski AK
    2. Wróblewska J
    Development of blastomeres of mouse eggs isolated at the 4- and 8-cell stage
    Journal of Embryology and Experimental Morphology 18:155–180.

Article and author information

Author details

  1. Nestor Saiz

    Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0637-791X
  2. Anna-Katerina Hadjantonakis

    Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States
    For correspondence
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7580-5124

Publication history

  1. Version of Record published: March 28, 2017 (version 1)


© 2017, Saiz et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.


  • 867
    Page views
  • 142
  • 0

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Nestor Saiz
  2. Anna-Katerina Hadjantonakis
Totipotency: A developmental insurance policy
eLife 6:e26260.

Further reading

    1. Developmental Biology
    Zain Alhashem et al.
    Research Article Updated

    Coordination of cell proliferation and migration is fundamental for life, and its dysregulation has catastrophic consequences, such as cancer. How cell cycle progression affects migration, and vice versa, remains largely unknown. We address these questions by combining in silico modelling and in vivo experimentation in the zebrafish trunk neural crest (TNC). TNC migrate collectively, forming chains with a leader cell directing the movement of trailing followers. We show that the acquisition of migratory identity is autonomously controlled by Notch signalling in TNC. High Notch activity defines leaders, while low Notch determines followers. Moreover, cell cycle progression is required for TNC migration and is regulated by Notch. Cells with low Notch activity stay longer in G1 and become followers, while leaders with high Notch activity quickly undergo G1/S transition and remain in S-phase longer. In conclusion, TNC migratory identities are defined through the interaction of Notch signalling and cell cycle progression.

    1. Developmental Biology
    2. Neuroscience
    Eleni Chrysostomou et al.
    Research Article

    Neurogenesis is the generation of neurons from stem cells, a process that is regulated by SoxB transcription factors (TFs) in many animals. Although the roles of these TFs are well understood in bilaterians, how their neural function evolved is unclear. Here, we use Hydractinia symbiolongicarpus, a member of the early-branching phylum Cnidaria, to provide insight into this question. Using a combination of mRNA in situ hybridization, transgenesis, gene knockdown, transcriptomics, and in-vivo imaging, we provide a comprehensive molecular and cellular analysis of neurogenesis during embryogenesis, homeostasis, and regeneration in this animal. We show that SoxB genes act sequentially at least in some cases. Stem cells expressing Piwi1 and Soxb1, which have a broad developmental potential, become neural progenitors that express Soxb2 before differentiating into mature neural cells. Knockdown of SoxB genes resulted in complex defects in embryonic neurogenesis. Hydractinia neural cells differentiate while migrating from the aboral to the oral end of the animal, but it is unclear whether migration per se or exposure to different microenvironments is the main driver of their fate determination. Our data constitute a rich resource for studies aiming at addressing this question, which is at the heart of understanding the origin and development of animal nervous systems.