Aberrant inhibitory morphogen decoding in the apical ectodermal ridge of regenerated limbs with recurrent patterning defects

Background Hypersensitivity to inhibitory cues is a core feature of appendages that repeatedly redevelop patterning errors. Such hypersensitivity is thought to arise from aberrant processing of negative-feedback signals. The apical ectodermal ridge (AER) is a principal locus for decoding this feedback during outgrowth. Although impaired inhibitory decoding in malformed regenerates is well recognised, it remains largely unknown whether this dysfunction and its signaling signature persist after apparently complete, scar-free regeneration. Methods Light-sheet fluorescence data from 36 drug-naïve adult salamanders with a history of recurrent digit duplication, yet currently displaying anatomically normal limbs, and 27 age-matched controls were recorded during a BMP7/Chordin pulse-conditioning paradigm. Within a biophysical modelling framework we quantified the temporal-error (ΔGrad) dependent activity of AER cells during inhibitory cue integration. Generalised morphogenetic interaction analyses were then used to characterise functional coupling of the ΔGrad signal with the underlying progress zone. Results Compared with controls, history-positive regenerates exhibited significantly elevated ΔGrad-linked inhibitory signaling in bilateral AER domains. The magnitude of this response correlated with subtle residual digit-length asymmetries in the same animals. In addition, these regenerates showed reduced functional connectivity between the AER and distal mesenchymal progenitors. Conclusions The heightened AER response to inhibitory morphogens—particularly during the expectation phase of growth arrest—together with weakened AER–progress zone connectivity in regenerated yet previously defective limbs, indicates a persistent hypersensitivity to, and/or impaired regulation of, anti-morphogenetic environmental cues during pattern restoration.