Growth factor-mediated coupling between lineage size and cell fate choice underlies robustness of mammalian development

Néstor Saiz, Laura Mora-Bitria, Shahadat Rahman, Hannah George, Jeremy P Herder, Jordi García-Ojalvo, Anna-Katerina Hadjantonakis

Posted on: 19 January 2020

Preprint posted on 27 December 2019

Article now published in eLife at

FGF4 signalling in the blastocyst: A simple system for an important binary cell fate decision.

Selected by Pierre Osteil, Irepan Salvador-Martinez

Categories: developmental biology


During the development of the vertebrate embryo, its cells must divide and specialise into different cell types. The first specialisation occurs when cells take a binary decision: to form the embryo body or its supporting tissues such as the placenta or the yolk sac. Despite decades of work, some mechanisms involved in this early transition remain poorly understood. It is unclear how the embryo controls the proportions of cells when proliferating. Especially given the observation that after modifying the ratio of progenitor cells, the proportions revert to normal after some time.

As a paradigm of this early cell fate decision in mammals, the team chose to address the preimplantation mouse embryo. The preimplantation embryo first multiplies the number of cells to form a mass (called morula) until the first specialisation: its internal cells become the inner cell mass (ICM) while the outer cells become the trophectoderm (TE; the progenitor cells of the placenta). The ICM then further specialises into the epiblast (Epi) and the primitive endoderm (PrE). The epiblast is comprised of the progenitors of all cells in the adult body, while the PrE cells contribute to both extraembryonic structures (the placenta and yolk sac) and the definitive endoderm (progenitors of the gastrointestinal tract).

This study was built upon two observations made during the development of mammalian embryos: the development of the embryo is not affected by 1) removing cells at early stages for preimplantation diagnosis in human and 2) adding cells in the mouse embryo during the generation of chimeras (by injecting embryonic stem cells – ESC) These findings led the team to the hypothesis that an internal signalling pathway could be controlling the cell fate and lineage sizes in the blastocyst, reflecting on its adaptability during early development.

The team previously showed that the three lineages forming the blastocyst (TE, Epiblast and PrE) are comprised of a consistent proportion of lineage-derived cells, suggesting a robust regulation of the cell population (Saiz et al. 2016). Previous studies have proposed that a direct mutual inhibition between NANOG (expressed in the ICM) and GATA6 (expressed in the PrE), could be responsible for the second stage of cell specialisation (Bessonnard et al. 2014; Huang et al. 2007; Nissen et al. 2017; Schröter et al. 2015; Tosenberger et al. 2017, 2019). However,  the expression of Gata6 is supported by the MAPK pathway which is activated by FGF4 but the epiblast fate is obtained from a low FGF environment (Chazaud et al. 2006). Taken together, these studies led the authors to the hypothesis that FGF4 alone, rather than a direct inhibitory circuit between NANOG and GATA6, could be responsible for the rapid cell fate switch between the two cell types during the second cell specialisation.

To test this hypothesis, the authors designed an in silico model for cell proportions and differentiation, generated chimeras to build up the cell number in the embryo and laser dissection to reduce it and finally demonstrated that FGF4-controlled local gradients are solely responsible for the progenitors to undergo specialisation into PrE or Epi.


The results:

Chimeras were formed by aggregating WT GFP+ embryonic stem cells (ESC) into label-free Gata6-/- embryos. As the PrE requires the expression of Gata6, the Gata6-/- host cells cannot form the PrE. However, when WT ESCs are injected into the embryo, they can specialise into PrE and rescue its development. Interestingly, the authors noticed the fate of WT cells depended on the final ratio of WT to Gata6-/- cells in the embryo. Indeed, if the injected WT cells accounted for less than or equal to 40% of the total cell number, WT cells only formed PrE. In contrast, if the WT cells accounted for more than 40% of the total number of cells, they contributed to both the epiblast and PrE. This data suggests a non-cell autonomous mechanism for cell fate decision, or, in simpler words, that cell fate is decided based on the cell neighbourhood activity.

To gain a better understanding of the robustness of the ICM fate decision in a WT scenario, the team aggregated increasing amounts of fluorescently-labelled WT ESCs with unlabelled WT host embryos, to evaluate the effects of the varying proportion of ESC- to host-derived EPI cells (from 0.5x to 8x the host EPI cells). The ESC injection was performed at the 8-cell morula stage, before the first specialisation of the host blastomeres into ICM and TE. Interestingly, the number of cells injected did not affect the number of host-derived ICM cells indicating a cell-autonomous differentiation. However, it did impact the cell fate from the second specialisation event of the ICM; the more ESCs added, the more the host embryonic cells shifted away from the epiblast towards a PrE fate, confirming that the second specialisation was not cell-autonomous and instead based on the ratio of cells in the blastocyst.




To gain a more precise spatiotemporal control of lineage size manipulation, Saiz and colleagues optimised laser cell ablation to eliminate any desired cell in the blastocyst. Using this method, they observed a striking recovery; even after ablating either all Epi or PrE cells, all embryos contained cells of both lineages after 16-20 hours. However, this ability was lost at later developmental stages demonstrating a decline in the adaptability of the embryo as it develops. It has been demonstrated that FGF4 is involved in ICM specialisation into PrE. Saiz and collaborators hypothesised that FGF4 is essential in the growth factor-mediated feedback they proposed in their minimal mathematical model. Supporting this hypothesis, Fgf4-/- embryos do not form PrE and can be rescued by injecting WT ESCs with normal Fgf4 expression. Interestingly, the size of the resulting PrE was directly proportional to the number of WT ESCs injected.

In summary, the authors propose that the ratio of Epi versus PrE cells – rather than the absolute cell number – is an important determinant for cell fate, and that the expression of FGF4 is enough to regulate the Epi versus PrE cell fate decision.


Why did I choose this article?

I chose this article for the quality of the experiments performed and the amount of new insights gleaned by the team. It shows how flexible (hence impressive) the mammalian embryo is but also its limitations, giving the reader a clearer idea of its adaptability spectrum: one must add or remove a lot of cells to prevent the embryo to further develop. Furthermore, the team showed local concentrations of FGF4 ligand alone provide a fast and simple cue for cell fate changes. This is further supported by a simple mathematical model established by the authors. I enjoyed the simplicity of this model to explain the binary cell fate decision.




Bessonnard, Sylvain, Laurane De Mot, Didier Gonze, Manon Barriol, Cynthia Dennis, Albert Goldbeter, Geneviève Dupont, and Claire Chazaud. 2014. ‘Gata6, Nanog and Erk Signaling Control Cell Fate in the Inner Cell Mass through a Tristable Regulatory Network’. Development (Cambridge) 141(19):3637–48.

Chazaud, Claire, Yojiro Yamanaka, Tony Pawson, and Janet Rossant. 2006. ‘Early Lineage Segregation between Epiblast and Primitive Endoderm in Mouse Blastocysts through the Grb2-MAPK Pathway’. Developmental Cell 10:615–624.

Huang, Sui, Yan Ping Guo, Gillian May, and Tariq Enver. 2007. ‘Bifurcation Dynamics in Lineage-Commitment in Bipotent Progenitor Cells’. Developmental Biology 305(2):695–713.

Menchero, Sergio, Isabel Rollan, Antonio Lopez-Izquierdo, Maria Jose Andreu, Julio Sainz De Aja, Minjung Kang, Javier Adan, Rui Benedito, Teresa Rayon, Anna Katerina Hadjantonakis, and Miguel Manzanares. 2019. ‘Transitions in Cell Potency during Early Mouse Development Are Driven by Notch’. ELife 8:1–29.

Nissen, Silas Boye, Marta Perera, Javier Martin Gonzalez, Sophie M. Morgani, Mogens H. Jensen, Kim Sneppen, Joshua M. Brickman, and Ala Trusina. 2017. ‘Four Simple Rules That Are Sufficient to Generate the Mammalian Blastocyst’. PLoS Biology 15(7):1–30.

Rayon, Teresa, Sergio Menchero, Andres Nieto, Panagiotis Xenopoulos, Miguel Crespo, Katie Cockburn, Susana Cañon, Hiroshi Sasaki, Anna Katerina Hadjantonakis, Jose Luis de la Pompa, Janet Rossant, and Miguel Manzanares. 2014. ‘Notch and Hippo Converge on Cdx2 to Specify the Trophectoderm Lineage in the Mouse Blastocyst’. Developmental Cell 30(4):410–22.

Saiz, Néstor, Kiah M. Williams, Venkatraman E. Seshan, and Anna Katerina Hadjantonakis. 2016. ‘Asynchronous Fate Decisions by Single Cells Collectively Ensure Consistent Lineage Composition in the Mouse Blastocyst’. Nature Communications 7.

Schröter, Christian, Pau Rué, Jonathan Peter Mackenzie, and Alfonso Martinez Arias. 2015. ‘FGF/MAPK Signaling Sets the Switching Threshold of a Bistable Circuit Controlling Cell Fate Decisions in Embryonic Stem Cells’. Development (Cambridge) 142(24):4205–16.

Tosenberger, Alen, Didier Gonze, Sylvain Bessonnard, Michel Cohen-Tannoudji, Claire Chazaud, and Geneviève Dupont. 2017. ‘A Multiscale Model of Early Cell Lineage Specification Including Cell Division’. Npj Systems Biology and Applications 3(1):1–10.

Tosenberger, Alen, Didier Gonze, Claire Chazaud, and Geneviève Dupont. 2019. ‘Computational Models for the Dynamics of Early Mouse Embryogenesis’. International Journal of Developmental Biology 63(3-4–5):131–42.






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Author's response

Anna-Katerina Hadjantonakis, Jordi García-Ojalvo, Néstor Saiz shared

Could you explain to us in simpler way how the mathematical model you designed is helping to understand the cell fate decision of the mouse embryos?

The model we propose here is a minimal model in which the element driving symmetry breaking in the ICM is the growth factor FGF4. Previously proposed models (both mathematical and conceptual) contained the growth factor, but also a second component, namely direct mutual inhibition between the transcription factors NANOG and GATA6. Our mathematical model allows us to isolate the effect of intercellular feedback via FGF, in the absence of direct intracellular mutual inhibition between NANOG and GATA6. It is important to point out that this decoupling is something that cannot be performed experimentally. The fact that such a model is able to qualitatively recapitulate the robust behaviour exhibited experimentally by the embryo suggests that FGF feedback is sufficient for the robustness of the cell fate decision taking place in the embryo.

How does the robustness explained by this model of indirect inhibition between Nanog and Gata6 would compare to a delta-notch lateral inhibition model?

One of the conclusions that can be extracted from our model is that the decision between Epi and PrE cells is driven by a mechanism akin to Delta-Notch lateral inhibition, even though, as far as we know, Delta-Notch signaling is not involved in this process. Interestingly, Notch signalling has been proposed to operate in the mouse preimplantation embryo, but in an earlier decision where cells decide between an ICM and trophectoderm fate (Menchero et al. 2019; Rayon et al. 2014) . The lack of involvement of Notch here is perhaps surprising, given that the salt-and-pepper distribution of epiblast and PrE cells in the early/mid blastocyst, first observed by Claire Chazaud, Yojiro Yamanaka and Janet Rossant (Chazaud et al. 2006), is reminiscent of the outcome of lateral inhibition. The difference between the two mechanisms is that in the case of Delta-Notch, cell-cell communication is mediated by membrane receptors and ligands acting in a juxtracrine manner, whereas in our case the interaction takes place via signalling molecules that diffuse across short distances in embryonic tissue. However, this short diffusion could create regions of high ligand concentration around the producing cells or cells that produce HSPGs (which potentiate FGF binding to their cognate receptors), as has been proposed by others, and thus have a similar effect to a membrane-bound ligand.

Given that FGF signalling pathway is widely used in developmental patterning, do you think its role in indirect inhibition via growth-factor has been co-opted?

It is highly likely that indirect inhibition via FGF4 has been contextually co-opted in rodent blastocysts to ensure the decision takes place robustly, despite the small number of cells and the time constraints imposed by implantation into the uterus. Current evidence from other mammalian species suggests FGF plays a role in this cell fate decision, but also that there are likely to be additional inputs. Namely, FGF is not necessary and sufficient in other mammals, as it is in mouse or rat embryos. The preimplantation period of these animals is longer and the embryos are larger (in terms of cell numbers), and those factors may help buffer noise in the patterning process.

What would be your approach to validate this mechanism in the human embryo? Using micropatterns? Blastoids?

Since they are synthetic embryo-like structures recapitulating aspects of preimplantation development, Blastoids would be an interesting system to try. However, it would be important to first compare the lineage composition (namely the numbers of cells assigned to each of the 3 lineages) of blastoids to that of blastocysts and, critically, determine how variable it is! Some of the tools we used in our study, like laser cell ablation, could potentially be directly applied to human blastocysts, but of course, it would not be possible to use as many embryos as we did, and with human embryos being larger, the procedures would be much more involved – let alone the ethical discussion to be had first.

You mentioned that after cell ablation you observed an increase in progenitor survival. Do you think some DP cells are maintained until both lineages are fully specified? Are these progenitors maintained by sensing the cell ratio between Epi and PrE?

Absolutely, this could very well be the case. We have not explored this avenue of investigation, but of course, progenitor cells could sense the concentration of a factor (be that FGF4 or something else) which abundance reflects the number of epiblast and PrE cells specified. It could also be possible that progenitor cells are normally generated in excess, perhaps representing an intrinsic fail-safe mechanism for robustness, with a fraction of them eventually being lost in the unperturbed embryo but maintained in this context. We do not have data from so many embryos though, so while we’re excited by what it may potentially be telling us, we are on the side of caution with respect to this observation.

In the discussion you mentioned that this model “may be generalizable to the formation of other self-organizing, autonomous developmental units”. Do you think a similar approach could apply for gastrulation of the mouse embryo? There are more lineages and signalling factors involved. What is your opinion on this? 

Gastrulation is a ‘holy grail’ for developmental biologists. It embodies the coordination of cell fate specification, cell movement and embryo size. Definitely a much more complex process, where at least 4 signals (as well as some of their inhibitors) converge on an ever-changing tissue-level landscape (Morgani et al., 2020). However, at its core, the basic model we propose could be adapted and deployed in other contexts, as a way to sense the number of cells acquiring distinct identities, to ensure control at the local level.

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