Morphogenetic event structure sculpts stem-cell population dynamics in the lateral epithelial compartment

INTRODUCTION Our experience of tissue regeneration unfolds as a stream of morphogenetic events that can later be reconstructed from stable pattern memory in striking detail. The limb field, critical for such episodic regeneration, has been shown to exhibit slow changes in cellular state over time, most prominently in the lateral epithelial compartment (LEC). It remains to be determined whether and how this drift in cell-population state contributes to the temporal organization of morphogenesis. RATIONALE Regenerative programmes are segmented into discrete events across a range of timescales from hours to days or more. Event boundaries—transitions between successive programmes such as wound closure, blastema consolidation, or differentiation waves—affect memory for the duration and order of events, which suggests that event structure could play an important role in shaping the cell dynamics underlying final pattern. To search for the mechanisms that determine how events are segmented and organized in time, we used high-density multiplexed voltage- and transcriptome-profiling arrays to record from an unprecedented number of cells in the LEC and neighbouring compartments in freely regenerating axolotl limbs, across multiple morphogenetic states, and in the presence of variations in event structure at multiple timescales. RESULTS Cell-population states in the LEC drifted continuously along a one-dimensional manifold during individual regenerative sessions, such that the ensemble trajectory travelled progressively farther away from the initial state. Simultaneously recorded states in the medial epithelial compartment (MEC) and the blastemal core (BC1) exhibited minimal drift. Recordings during nocturnal quiescence revealed that LEC dynamics were nearly identical to those observed during active regeneration, suggesting that drift does not require changing external cues and instead reflects an inherent network phenomenon. During active phases, population trajectories abruptly shifted at event boundaries, leading to the segmentation of cellular activity into discrete temporal units. During engineered protocols with repeating temporal structure, the LEC simultaneously encoded event information across multiple timescales by travelling additionally in directions orthogonal to the drift. We uncovered potential mechanisms for both drifting and shifting dynamics. Drift could be explained by hour-scale variability in the transcriptional and ion-channel expression of individual cells broadly distributed throughout the LEC. These slow fluctuations were necessary and sufficient for drift at the population level. Shifts at event boundaries were driven by synchronous bioelectrical and calcium transients across distinct cell ensembles in the LEC. Different ensembles responded at different boundaries, such that individual events could be time-stamped in pattern memory. CONCLUSION Drift of cellular state in the LEC is an inherent phenomenon that continues at a constant rate during both quiescent and active phases but is briefly interrupted by abrupt shifts at moments of transition between regenerative events. These results identify a candidate mechanism for the segmentation of morphogenetic experience into discrete units, as reported in human tissue studies. Regeneration consists of a sequence of events across a wide range of timescales, organized hierarchically from hours to days or more. We show that LEC dynamics simultaneously encode event information across these different timescales without exogenous reinforcement or extensive bioengineering. Together, our results identify a hierarchical coding scheme for organizing morphogenetic events in time.