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

## TL;DR

This study reveals how male and female Chinese mitten crabs regenerate claws differently at the molecular level, with key differences emerging early in the process.

## Contribution

The study identifies the papilla stage as a critical molecular transition and highlights the role of Antp and sex-regulatory networks in cheliped regeneration.

## Key findings

- Sexual dimorphism in cheliped regeneration is established at the molecular level as early as 4 days post autotomy.
- The Hox gene Antp is a master regulator of appendage patterning and exoskeleton assembly during regeneration.
- RNAi knockdown of Antp disrupts joint differentiation and confirms its regulatory role in regeneration.

## Abstract

The ability to regenerate chelipeds is vital for the survival of the Chinese mitten crab (Eriocheir sinensis), yet the molecular mechanisms driving sexual dimorphism during this process remain unclear. This study combined morphological and transcriptomic analyses to investigate regenerative stages and sex-related differences. We found that while physical differences, such as larger regenerating chelipeds in males, only become prominent at 28 days post autotomy (dpa), the molecular foundation for this divergence is established as early as 4 dpa. This early divergence is driven by sex-specific endocrine networks, with males and females showing distinct gene expression patterns and hormonal activities. Furthermore, we identified the Hox gene Antennapedia (Antp) as a master regulator. RNAi-mediated knockdown of Antp resulted in impaired joint differentiation and confirmed its role in orchestrating appendage patterning and exoskeleton assembly. These findings suggest a putative hierarchical regulatory model, wherein steroid hormones may enter regenerative tissue cells and bind to their corresponding hormone receptors, which could in turn regulate the expression of developmental genes such as Antp. Such regulation might potentially modulate downstream transcriptional programs and contribute to the structured regeneration of limbs, providing novel speculative insights into crustacean developmental biology.

Cheliped regeneration in the E. sinensis 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 Fem-1c-like, Cyp2L1-like, CpAMP1A-like and Nedd4-like, while females showed enrichment in elevated aromatase activity. Weighted gene co-expression network analysis (WGCNA) identified the Hox gene Antp as a core hub regulator, exhibiting high co-expression with key epidermal-related genes such as Cht6, Cht2-like 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 Antp resulted in obscured differentiation between the propodus and carpus in both sexes and confirmed its regulatory control over downstream targets including Ubx, Bmp2-like, and CpAMP1A-like. This study suggests a putative hierarchical regulatory model in which systemic hormonal signals may integrate Antp 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.