The Papilla Stage as a Critical Molecular Transition: Antp and Sex-Regulatory Network Orchestrate Cheliped Regeneration in Eriocheir sinensis

Cheliped regeneration in the <i>E. sinensis</i> is a tightly regulated physiological process, yet the molecular regulatory mechanisms underlying sexual dimorphism during regeneration remain unclear. In this study, we combined morphological observation with transcriptomic analysis to systematically investigate the regenerative stage characteristics and sex-related differences. The papilla stage 4 dpa was identified as a pivotal transitional stage, bridging initial wound healing and cellular dedifferentiation (2 dpa) with subsequent redifferentiation and morphogenesis (7 dpa). Morphological sex-based differences characterized by larger regenerating chelipeds in males became prominent by the late stage (28 dpa). Notably, the molecular foundation of sexual dimorphism was found to be established at 4 dpa, significantly preceding the emergence of phenotypic differences. This early divergence was driven by sex-dimorphic endocrine networks: males exhibited preferential expression of genes such as <i>Fem-1c-like</i>, <i>Cyp2L1-like</i>, <i>CpAMP1A-like</i> and <i>Nedd4-like</i>, while females showed enrichment in elevated aromatase activity. Weighted gene co-expression network analysis (WGCNA) identified the Hox gene <i>Antp</i> as a core hub regulator, exhibiting high co-expression with key epidermal-related genes such as <i>Cht6</i>, <i>Cht2-like</i> and more. Its suppressed expression at 2 dpa aligned with the requirements for dedifferentiation, whereas its peak at 4 dpa indicated a crucial role in orchestrating appendage patterning and exoskeleton assembly. RNA interference (RNAi) knockdown of <i>Antp</i> resulted in obscured differentiation between the propodus and carpus in both sexes and confirmed its regulatory control over downstream targets including <i>Ubx</i>, <i>Bmp2-like</i>, and <i>CpAMP1A-like</i>. This study suggests a putative hierarchical regulatory model in which systemic hormonal signals may integrate <i>Antp</i> and other sex-biased regulators to potentially facilitate structured limb regeneration. These findings offer tentative novel insights into the interplay between developmental plasticity and sex-based regulatory divergence in decapod crustaceans.