Epigenetic pattern memory increasingly guides appendage regeneration as bioelectric cue sensitivity wanes with ontogenetic maturation

The capacity of multicellular collectives to re-establish correct organ form after injury undergoes a prolonged refinement during larval and juvenile stages, yet the mechanisms that drive the emergence of high-fidelity pattern repair remain elusive. In a series of experiments performed on the same cohort of stage-matched amphibian hatchlings through late metamorphic larvae, we examined how spontaneous bioelectric discrimination of positional gradients and activation of epigenetic pattern memory become integrated across ontogeny. Bioelectric discrimination was quantified with a voltage-sensitive dye frequency-tagging paradigm (Study 1). Pattern memory was assessed via chromatin-state similarity profiling using CUT&RUN for histone modifications associated with positional identity (Study 2). Two functional assays—blastema cell sorting and proximodistal graft matching—were used to evaluate regenerative accuracy (Study 3). Representational similarity analysis determined the predictive influence of bioelectric and epigenetic metrics on morphological outcomes. We show that, although the ability to resolve canonical proximal-distal voltage gradients is present during early larval stages, its contribution to regeneration fidelity diminishes with maturation. Conversely, successful inference of target morphology increasingly depends on epigenetic pattern memory as organisms age and accumulate morphogenetic experience.