Brazil Nut Effect Drives Pattern Formation in Early Mammalian Embryos

Zheng Guo, Jie Yao, Xu Zheng, Jialing Cao, Zheng Gao, Shuyu Guo, Dandan Qin, Min Tan, Bo Wang, Fanzhe Meng, Jing Zhang, Lei Li, Jing Du, Yubo Fan

Posted on: 5 April 2021 , updated on: 21 April 2021

Preprint posted on 7 March 2021

Shake, rattle, and roll: in their latest preprint, Zheng Guo et al show how vibrations of the entire blastocyst cavity drive one of the earliest mammalian patterning events.

Selected by Sophia Friesen

Background and context:

All the complexity of multicellular organisms arises from a single, often unpatterned cell. How this happens is the central question of developmental biology, and often, the answer has to do with chemical signaling gradients. From the veins of a fly’s wing to the fingers of a human hand, chemical gradients provide a “coordinate system” for tissue patterning. But the first few steps of mammalian differentiation and patterning occur without known chemical gradients. Here, Guo et al. asked whether mechanical forces could drive the second patterning event in mammalian development, by carefully observing tissue movements in early mouse embryos and by pharmacologically changing their mechanical properties.

The first two cell fate decisions in mammalian embryos distinguish cells that will become the embryo itself from various extra-embryonic support tissues. First, trophectoderm (TE) cells, which will become part of the placenta, are distinguished from inner cell mass (ICM) cells, and sort to the outside of the spherical embryo. TE cells then pump ions into the center of the embryo, and water follows the ions, so that a high-pressure, fluid-filled cavity forms.

As the blastocyst cavity grows larger, a second round of differentiation and patterning occurs. ICM cells become either epiblast cells (EPI), which form the fetus, or primitive endoderm (PrE), which develops into a layer of extraembryonic tissue. At first, these two cell types are mixed together, seemingly randomly, but then sort out so that PrE cells are next to the blastocyst cavity and EPI cells are next to TE. Guo et al. investigated a surprising link between EPI/PrE sorting and the mechanical properties of the blastocyst cavity.


Key findings:

  1. The entire blastocyst cavity vibrates, which correlates with migration of PrE cells

The researchers began by live imaging the entire course of PrE cell migration, from before PrE and EPI cells differentiated until the two cell types sorted out completely. Over this time period, the blastocyst cavity grows and then actually starts vibrating, with periods of rapid shrinking followed by longer, slower periods of growth [1]. These oscillations are caused by cell divisions within the TE, which briefly disrupt tissue integrity, causing the high-pressure blastocyst to “deflate” slightly before growing again [2]. Intriguingly, periods of cavity shrinking seemed to correlate with faster movement of PrE cells towards the cavity.

The researchers proposed that the vibration of the blastocyst cavity could cause PrE cells to sort towards the cavity. PrE and EPI cells may have different adhesive properties [3], which would help separate the cell types, while the rapid shrinking and slower growth periods of the vibration could provide the directional force needed to correctly orient the two cell populations. In fact, non-adhesive plastic beads injected into the ICM could also sort out to the interface with the cavity! This shows that differential adhesion combined with global tissue movement is enough to explain spatial sorting of cell populations.

  1. Altering blastocyst vibrations by pharmacologically changing cavity pressure can speed up or slow down EPI/PrE specification and sorting

To figure out whether blastocyst vibrations were required for PrE cell sorting, the researchers first grew blastocysts in hypertonic media, which depressurizes the blastocyst cavity and dampens its vibrations. In this condition, EPI/PrE sorting is delayed, and so is the specification of distinct EPI and PrE populations, suggesting that blastocyst vibration is important not only for PrE migration but for differentiation as well. The high-pressure, hypertonic blastocyst cavity is created by action of the Na+/H+ pump, and using a drug to block this pump likewise interfered with EPI/PrE specification and sorting.

These results were promising, but not conclusive – perhaps the affected embryos were just generally sick. But by increasing cavity pressure and vibration frequency, either by exposing embryos to hypotonic media or by pharmacologically boosting Na+/H+ pump activity, the researchers were actually able to speed up EPI/PrE specification and sorting to faster than normal. This provided clear evidence that mechanical forces from the blastocyst cavity drive specification of EPI and PrE cells as well as their spatial segregation.


  1. YAP, a mechanically sensitive transcription factor, is required for EPI/PrE specification and sorting

The researchers suspected that the transcription factor YAP might be involved in EPI/PrE specification and sorting. YAP can upregulate one of the earliest markers of PrE cells, at least in some contexts, and YAP activity is affected by mechanical forces, making it a promising candidate to connect blastocyst vibration to EPI/PrE specification. As EPI and PrE cells differentiate, the researchers observed that YAP became more nuclear in PrE cells compared to EPI cells, indicating stronger YAP activity. Increasing cavity pressure and vibration speed by exposing cells to hypotonic media also makes YAP more nuclear, confirming that YAP is responsive to mechanical forces in this context. Finally, pharmacologically blocking YAP activity decreases the number of ICM cells that specify to either PrE or EPI cells, and prevents those cells from spatially sorting out.


Why I liked this paper:

A classic research strategy to try to understand a biological process is to figure out a way to break it. Making processes run faster, as the researchers did by increasing blastocyst pressure and vibration frequency, is unusual and very impressive. Similarly, showing that spatial segregation can operate even on biologically inert plastic beads, based only on the physical mechanics of the tissue around them, is a fascinating result!

While signalling gradients are foundational to developmental patterning, the role of mechanical forces is increasingly being appreciated. Researchers are finding more and more contexts in which tension, compression, and stiffness influence development, and this is a particularly cool example that links a key mammalian patterning event to a dramatic tissue movement. The same basic process that brings the biggest cereal flakes to the top of the box also sorts cells into distinct populations – who knew?

Image credit: modified from original by Berichard; license available here.


Questions for the authors:

  1. Most of the manipulations you perform affect blastocyst vibration by changing cavity pressure. What’s your best evidence that vibration itself, rather than cavity pressure, drives EPI/PrE specification and tissue movements?
  2. It’s intriguing that blocking YAP activity decreases the proportion of EPI cells, when decreased YAP activity is associated with EPI cell fate during normal development. Why doesn’t verteporfin-treated ICM develop into 100% EPI cells?
  3. As you mention yourselves, drug-based manipulation of blastocysts is limited, and could be affecting all cells of the embryo in unexpected ways. If you decided to genetically manipulate blastocysts to continue this research, what’s the first experiment you would do?



  1. Niimura S. (2003). Time-lapse videomicrographic analyses of contractions in mouse blastocysts. J Reprod Dev doi:10.1262/jrd.49.413
  2. Chan CJ, Costanzo M, Ruiz-Herrero T et al. (2019). Hydraulic control of mammalian embryo size and cell fate. Nature 571, 112-116.
  3. Plusa B, Piliszek A, Frankenberg S, et al. (2008). Distinct sequential cell behaviours direct primitive endoderm formation in the mouse blastocyst. Development 135, 3081-3091. doi: 10.1242/dev.021519

Tags: blastocyst, development, epiblast, mammal, mechanics, mouse, primitive endoderm, vibration


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Author's response

Jing Du shared

Actually, the research in the cross-field of biophysics is very meaningful and challenging. I am glad to answer your questions.

  1. Most of the manipulations you perform affect blastocyst vibration by changing cavity pressure. What’s your best evidence that vibration itself, rather than cavity pressure, drives EPI/PrE specification and tissue movements?

The vibration and pressure of the blastocyst cavity are accompanied by each other, and there are few methods to study the two independently. In our experiments, the blastocyst vibration was also suppressed by inhibiting the division of TE cells with aphidicolin without affecting the cavity pressure. The results showed that ICM fluidity and PrE precursors movement were both significantly reduced by aphidicolin-treatment. These results indicate that the increase in cavity pressure alone without blastocyst vibration is not enough to drive EPI/PrE segregation.

  1. It’s intriguing that blocking YAP activity decreases the proportion of EPI cells, when decreased YAP activity is associated with EPI cell fate during normal development. Why doesn’t verteporfin-treated ICM develop into 100% EPI cells?

This is an interesting question. Although we did not have direct evidence, we could provide several assumptions regarding this problem: 1. The concentration of verteporfin we used to treat the embryos was moderate which may not blocked the entire activity of YAP. 2. The gene expression difference between EPI/PrE precursors already occurs at E3.5, while the YAP signal is very low in ICM and comparable between these two types of cells. This means that the YAP signaling pathway may function at later stage of lineage specification when the difference in gene expression between EPI/PrE was amplified. Unlike in the first cell fate decision, that YAP directly regulates the expression of CDX2 in TE cells, there is no evidence that YAP could directly regulates the marker gene expression of EPI or PrE. However, there is evidence that YAP signaling increases the expression of FGF receptors (FGFRs) in embryonic neural stem cells and lung cancer samples. Thus, we hypothesize that the YAP signal may promote the specificity of EPI/PrE through affecting FGF pathway. We plan to design experiments for yap signal pathway related problems in the future, and there will be follow-up work next.

  1. As you mention yourselves, drug-based manipulation of blastocysts is limited, and could be affecting all cells of the embryo in unexpected ways. If you decided to genetically manipulate blastocysts to continue this research, what’s the first experiment you would do?

As far as we know, there is few method including genetic manipulation that can successfully regulate gene expression specifically in time and space at the same time. According to the mechanism of “Brazil nut effect”, the completion of segregation is dependent on two properties: the cavity vibration and the difference in cohesiveness or motility between the two types of cells. Although it is difficult to find genetic methods that specifically regulate cavity vibration, it is possible to affect cohesiveness and motility. Actually, to further support our model, we are performing experiments to interrupt the adhesive and motile heterogeneity between EPI and Pre cells, such as interfering the expression of intercellular adhesion molecules (e.g., E-cadherin) or cell mechanics (such as cell surface tension regulating proteins, ERM) specifically by genetic strategies. We will also try to achieve other methods for regulating blastocysts spatiotemporally.

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