During pre-implantation development in mammalian embryos, compaction is the first of several key morphogenetic movements, marking the transition from loosely associated cells to a tightly organized structure that is essential for blastocyst formation. In the mouse, compaction occurs at the 8-cell stage and requires the cell-cell adhesion molecule Cdh1 (E-cadherin). At this stage, the embryo spreads its cell-cell contacts and reduces the surface exposed to external medium.

Changes in adhesion molecules like Cdh1 were initially thought to drive compaction by increasing adhesion energy, similar to soap bubbles. Yet, Cdh1 alone actually provides insufficient adhesion force to fully account for compaction. (see Maitre, J.-L. et al. and Chan, E.H. et al.)  Instead, actomyosin contractility plays a key role by increasing surface tension at the cell-medium interface, which helps pull cells together. As cell-cell contacts grow, mechanical coupling between cells strengthens, and cadherins can reorganize into adhesion rings that act as scaffolds for actomyosin forces to pull on, ultimately strengthening cell-cell adhesion.

Although the mechanics of cell-cell contact spreading are well characterized, how mechanical coupling changes during compaction remains less clear. In their recent study, “Mechanical strengthening of cell-cell adhesion during mouse embryo compaction,” published in the March 18, 2025, issue of Biophysical Journal, de Plater et al. address this gap. By combining dual pipette aspiration (DPA) with genetic manipulation of adhesion molecules, the authors measure rupture forces in an ex vivo reduced system of cell doublets, shedding light on mechanical coupling during compaction.

The approach

To study compaction in mouse embryos, de Plater and colleagues simplified this system into a minimal adhesive model: cell doublets. They dissociate mouse embryos at the 4-cell stage, allow them to divide, and obtain pairs of sister blastomeres at the 8-cell stage. These doublets mimic the behavior of cells in intact embryos during compaction and offer several advantages: they provide a controlled, reproducible geometry; allow precise timing of contact formation; and enable quantitative measurements of adhesive dynamics.

Cell doublets can be mechanically separated to directly measure (single) cell-cell adhesion coupling forces. To do this, the authors used DPA, a technique that physically pulls cells apart while measuring the forces required to rupture their contact—often reaching up to hundreds of nanonewtons. In this study, doublets were separated at different time points after divisions, binned into 2-h intervals, to measure separation forces over time.

Key findings

Using DPA, the authors found that contact angles and radii increased steadily throughout the eight-cell stage, with an increase in force required to separate doublets (separation force, Fs) from 40 to 70 nN, suggesting that mechanical stability increases as compaction progresses. Strong correlations between Fs and both contact angle and contact length suggest that growth of cell-cell contact area contributes significantly to the strengthening of cell-cell contacts during compaction.

How do cell-cell adhesions strengthen during compaction?

Although cell contacts expand, increased size alone may not fully explain increased mechanical stability of cell-cell contacts during compaction. To explore this further, the authors immunostained for Cdh1 and filamentous actin on doublets of increasing age to examine their molecular organization. As contacts grew, although Cdh1 levels remained relatively constant overall but became progressively enriched at the periphery, forming a ring-like structure at the contact edges, actin levels at contacts decreased. This suggests that mechanical stability may be associated with reorganization rather than clustering of adhesion molecules. Suprisingly, when Fs was normalized to the radius of contact (Rc)—a measure of rupture tension (Fs/Rc) that may reflect effective bond density—they found no change in rupture tension over time. Thus, enrichment of Cdh1 at the contact rim does not enhance mechanical stability on its own, and increased contact size remains the primary driver of adhesion strengthening during compaction.

Digging (surface-)deep: The role of Cdh1

So, how crucial is Cdh1 for mechanical strength during compaction? To address this question, de Plater et al. examined embryos lacking maternal Cdh1 (mCdh1^+/– embryos), which should have reduced Cdh1 levels at the time of compaction. Doublets (mCdh1^+/– doublets) derived from these embryos started with smaller contacts and were also mechanically weaker—measured by lower Fs —than wild-type (WT) doublets. Despite this, mutant doublets were still able to increase their contact size during the 8-cell stage.

Interestingly, mCdh1^+/– doublets exhibited higher rupture tension than did WT doublets at the beginning of the 8-cell stage, suggesting a higher effective bond density, but this tension eventually decreased to WT levels. This suggests that mutants probably make up for lower Cdh1 levels, but this compensatory effect diminishes as contacts enlarge.

Next, the authors investigated the role of altered molecular organization in mCdh1^+/– doublets by immunostaining mutant doublets. As expected, these had less Cdh1 at cell-cell contacts; yet, they still formed normal adhesion rings. Thus, the smaller size or altered organization of Cdh1 alone cannot adequately account for the reduced mechanical strength observed in mutant doublets.

In fact, the authors found that intracellular mechanical coupling to the actin cytoskeleton plays an essential role here. To test this, they engineered embryos expressing a chimeric cadherin, with the extracellular and transmembrane domains of Cdh1 but the intracellular domain of Cdh2. Despite having an integral adhesive interface, these doublets failed to increase their contact size. Thus, the intracellular domain of Cdh1 is probably crucial for cytoskeletal anchoring and mechanical coupling during compaction.

Conclusions

The study by de Plater et al. delves into the mechanics of compaction, a developmentally regulated adhesion process, by using a minimal adhesive system of cell doublets. Their work emphasizes how the mechanical stability of cell-cell contacts increases during this process, mostly via expansion of contact areas between cells rather than by strengthening individual molecular bonds.

Collectively, the findings of this study highlight the key roles of contact spreading, reorganization of adhesive molecules, and, importantly, how these molecules are tethered to the actin cytoskeleton. Although Cdh1 is necessary, de Plater et al. show that its role in compaction is more dependent on its intracellular signaling and coupling to the actin cytoskeleton than it is on how much of it is present or where it is recruited.

Notably, although compaction is not essential to mouse development, the authors highlight that it may still serve a significant mechanical role in preventing cells from detaching from the embryo. Compaction may therefore help hold the embryo together during its first important morphogenetic transformation during development.