The aberrant migration and differentiation of neural crest cells are associated with the production of HVP in bearded chicken.

Hyperpigmentation of the visceral peritoneum (HVP) is a sex-biased phenotype that reduces carcass value in yellow-feathered broilers. While GWAS studies have identified candidate loci, the cellular origin and molecular mechanisms driving HVP remain unknown. This study employed single-cell RNA sequencing to profile black, faded, and normal peritoneal tissues from bearded chickens at 40 and 120 d of age, functionally validating findings with fibroblast lines and primary melanocytes. We identified nine cell types, with melanocytes significantly elevated in HVP tissue. UMAP visualization shows nearly overlapping melanocyte and Schwann-cell clusters, suggesting that HVP melanocytes may share a common origin with Schwann cells along the ventromedial migratory pathway of neural-crest cells (NCCs). Aberrant melanocyte aggregation and migration drive HVP pathogenesis. CellChat analysis demonstrated pivotal roles for the SEMA3C-PLXND1 axis, which directs NCCs migration, and the TNC-SDC1 axis, which enhances melanocytes adhesion, in fibroblasts-melanocytes crosstalk. Transcription factor analysis highlighted HOX family regulation of NCCs to melanocytes differentiation. Experimentally, Downregulation of SDC1 reduced melanin synthesis but significantly enhanced the migratory capacity of melanocytes, while estradiol promoted melanogenesis and modulated extracellular matrix. HVP development involves four interconnected mechanisms: RA-HOX-mediated aberrant NCCs differentiation, SEMA3C-PLXND1-driven melanocyte barrier breaching, TNC-SDC1-mediated peritoneal colonization, and estrogen-coordinated melanin deposition. These findings provide a theoretical framework for breeding against HVP in broilers.