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

Background:

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.

 

 

References:

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. https://dev.biologists.org/content/141/19/3637.long

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. https://www.sciencedirect.com/science/article/pii/S1534580706001250?via%3Dihub

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. https://www.sciencedirect.com/science/article/pii/S0012160607001674?via%3Dihub

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. https://elifesciences.org/articles/42930

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. https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.2000737

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. https://www.sciencedirect.com/science/article/pii/S1534580714004080?via%3Dihub

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. https://www.nature.com/articles/ncomms13463

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. https://dev.biologists.org/content/142/24/4205

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. http://dx.doi.org/10.1038/s41540-017-0017-0

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. http://www.ijdb.ehu.es/web/paper/180418gd/computational-models-for-the-dynamics-of-early-mouse-embryogenesis

 

 

 

 

doi: https://doi.org/10.1242/prelights.16272

Read preprint (No Ratings Yet)