Phantom Positional Signals Modulate Canonical Patterning Versus Experimental Regeneration Pathways: A Longitudinal Analysis in Multicellular Organoids

Phantom positional signals (PPSs) denote spurious spatial cues that arise within developing tissues and can redirect morphogenesis toward sub-optimal trajectories. Analogous to mis-reading a morphogen gradient, PPSs may encourage cell assemblies to engage unvalidated regenerative programs rather than the empirically optimised pathways encoded by conserved morphogen cascades. Yet, inconsistent quantification of PPSs has obscured their formal characterisation. Here we introduce the Phantom Signal Index (PSI), a three-item bioelectric–transcriptomic assay designed to capture the prevalence of PPSs in real time. We tested its predictive value for two competing outcomes: (i) adherence to canonical patterning behaviours—sequential Hox activation, planar-cell-polarity alignment, and basement-membrane remodelling—and (ii) recruitment of experimental electroceutic cocktails (EECs) that bypass conventional morphogen signalling. Additional constructs included lineage-autonomous control (LAC), evolutionary pathway fidelity (EPF), and exogenous small-molecule responsiveness (ESMR). A cohort of 1 507 vertebrate limb-bud organoids (734 XY lines, 768 XX lines, 5 XO variants) was tracked across three developmental intervals. Structural equation modelling revealed that elevated PSI scores aligned with increased EEC utilisation and reduced compliance with canonical patterning. Crucially, LAC and EPF partially attenuated this link, forecasting greater engagement with conserved pathways and higher morphogen sensitivity. In contrast, heightened ESMR amplified the association between PPSs and EEC uptake. These findings furnish initial evidence that phantom positional information meaningfully shapes regenerative decision-making in multicellular systems. The PSI offers a tractable framework for future work dissecting how spurious spatial cues interact with intrinsic control networks to determine developmental fidelity and regenerative outcome.