Spatiotemporal antagonism between MAPK/ERK and BMP/SMAD1 signaling defines embryonic fate patterning in human stem cell colonies

Olivier Pertz

ERK signaling plays a central role in both pluripotency maintenance and differentiation of human embryonic stem cells (hESCs). These processes are inherently heterogeneous: under self-renewal conditions, some cells spontaneously begin to differentiate, while others fail to fully respond to differentiation cues, retaining self-renewal features. This functional variability complicates efforts to define signaling determinants of cell fate. We hypothesized that such heterogeneity might be governed by dynamic features of ERK activity at the single-cell level. To investigate this, we employed live-cell biosensor imaging to analyze ERK signaling dynamics in individual cells within 2D hESC colonies.

In colonies maintained under standard stem cell renewal conditions with FGF2, we discovered a robust and spatially organized pattern of ERK activity that pointed beyond cell-intrinsic fate regulation toward a role for ERK dynamics in spatially coordinating multicellular behavior. ERK activity was not homogeneous but formed a peripheral band of sustained signaling with a sharply defined boundary to a central zone of low or pulsatile ERK activity. This ERK ON band maintained a constant width across colony sizes and correlated with increased cortical myosin-based contractility at the colony edge. Notably, these ERK domains did not correspond to differences in proliferation or pluripotency marker expression, indicating that ERK dynamics in this context act independently of cell fate and instead shape the colony’s mechanical architecture.

To determine the origin of this spatial pattern, we manipulated FGF2 levels. Colonies deprived of FGF2 lost the peripheral ERK band, while reintroduction of FGF2 restored and reshaped the ERK activity profile. Low concentrations reinstated the peripheral ERK band, whereas high concentrations suppressed ERK signaling at both the edge and center, generating a ring-like ERK-high zone. This pattern arises from a peripheral-to-central FGF diffusion gradient, enabled by tight junction–mediated insulation of the colony’s dorsal surface. This apical sealing traps FGF within the basolateral space, forming a lateral gradient that is interpreted into sharp intracellular activity domains by the MAPK network downstream of the FGF receptor.

We next explored how this ERK architecture interacts with BMP4-mediated differentiation signals. Addition of BMP4 induces a peripheral SMAD1 signaling domain that spatially antagonizes ERK activity. This antagonism organizes the colony into three discrete zones: (1) a peripheral SMAD-high, ERK-low mesenchymal-like region; (2) a ring of SMAD-low, ERK-high epithelial cells; and (3) a central domain characterized by low SMAD signaling and stochastic ERK pulses. These signaling domains align with differential expression of early lineage markers: extraembryonic markers at the periphery, mesoderm and endoderm adjacent to the ERK-high ring, and ectodermal markers in the center.

These results demonstrate that spatially patterned ERK and SMAD signaling can emerge in unconfined hESC colonies through simple principles of ligand diffusion, epithelial sealing, and signaling network logic. This minimal yet robust system models key features of early human embryonic organization and offers a tractable framework for understanding how dynamic signaling encodes spatial fate information.