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. 2025 Apr 3;104(6):105114. doi: 10.1016/j.psj.2025.105114

Wnt signaling pathway and retinoic acid signaling pathway involved in delamination and migration of chicken trunk NCCs and contributing to HVP phenotype☆

Zhengyang Chen a,b,c, Changbin Zhao a,b,c, Rong Fu a,b,c, Chengyue Yuan a,b,c, Ke Zhang a,b,c, Xiquan Zhang a,b,c,⁎
PMCID: PMC12005351  PMID: 40209469

Abstract

Hyperpigmentation of the visceral peritoneum (HVP) is a hereditary trait that significantly affects the carcass quality in bearded chickens, yet its molecular mechanisms remain unclear. This study utilized data-independent acquisition proteomics to analyze the protein expression profiles of black peritoneum (B), faded peritoneum (F), and normal peritoneum (N) in bearded chickens at 40 and 120 d of age. Combined with histopathological and functional enrichment analyses, we revealed the regulatory network underlying HVP formation. Results indicated that the melanin content was significantly elevated in HVP samples, without accompanying inflammatory responses or tumor characteristics, suggesting that its formation is driven by developmental abnormalities. A total of 9,375 high-confidence proteins were identified through proteomics, with differentially abundant proteins at 40 d of age (219 proteins) primarily enriched in ribosomal function, tyrosine metabolism, and melanin synthesis pathways. In comparison, at 120 d of age (246 proteins), they were enriched in transcription regulation and chromatin remodeling pathways. The abnormal expression of key co-expressed proteins DHRS3 and DACT1 suggests that the dysregulation of retinoic acid (RA) and the Wnt signaling pathway may promote the directed differentiation of melanocytes by regulating neural crest cells (NCCs). The reduced abundance of the chondroitin sulfate proteoglycan, VCAN, weakened the peritoneal barrier function, whereas estradiol accelerated melanin synthesis via hormonal microenvironmental regulation. Furthermore, the formation of HVP led to a reprogramming of energy metabolism, reduced fat deposition, and a downregulation of immune-related molecules, implying that pigment deposition may weaken the chicken immune response. This study systematically elucidates the molecular mechanisms of HVP and provides potential targets for molecular breeding of HVP.

keywords: Hyperpigmentation of the visceral peritoneum, Wnt signaling pathway, Extracellular matrix, Neural crest cells

Introduction

Hyperpigmented Visceral Peritoneum (HVP) is a common quantitative trait in broiler chicken breeds such as bearded chickens(Luo et al., 2013). It is characterized by the abnormal aggregation of melanocytes in the connective tissue of the peritoneum and the deposition of melanin, although a small amount can also be found in other areas such as the neck, wings, and shanks. This condition is relatively common in amphibians, fish, and humans as well (Franco-Belussi et al., 2012; Uehara et al., 2019). At 40 d of age, the HVP characteristics of bearded chickens can be preliminarily observed through the abdominal skin. By 120 d of age, when the bearded chickens are ready for slaughter, the HVP features become very pronounced and significantly affect sales. Among 900 bearded chickens at 120 d of age, the proportion exhibiting HVP was as high as 37.25 % (unpublished data). Although there is no literature showing health problems caused by eating pigmented meat, the presence of HVP significantly affects the yield and quality of yellow-feathered broiler carcasses (Wang et al., 2024). It is worth noting that the formation of HVP is primarily governed by genetic factors (heritability score of 0.33), while also being co-regulated by environmental factors such as photoperiod, dietary composition, and sex (Moore and Smyth, 1971; Franco-Belussi et al., 2017). However, the molecular mechanisms remain unclear, particularly the relationship between developmental regulation abnormalities and the directional differentiation of melanocytes urgently requires further exploration.

Researchers have not isolated any pathogenic bacteria from HVP, nor have they observed significant inflammatory responses or tumor formation(Crespo and Pizarro, 2006). Electron microscopy of HVP only revealed the aggregation of melanocytes and the accumulation of melanin(Wang et al., 2014). Avian HVP is similar to excessive melanin deposition in the intestines, mesentery, and peritoneum of humans with gastrointestinal diseases. Through pathological observation and immunohistochemical methods, researchers have found that dysfunctional stromal cells (Kim et al., 2010; Sim et al., 2021), macrophages(Cappell et al., 2010; Lim et al., 2012), and neural crest cells (NCCs) (Jung et al., 1996) in the human peritoneum can lead to excessive melanin deposition, suggesting that avian HVP may be driven by developmental regulatory abnormalities rather than disease. The deposition of melanin in the peritoneal region may help protect visceral organs from the genotoxic effects of ultraviolet radiation and assist the body in adapting to larger temperature fluctuations (Franco-Belussi et al., 2017; Griffing et al., 2020). A genome-wide association study identified seven key genes, MFNG, CYP2D6, MYH9, and RANGAP1 (Zhou et al., 2022), as well as BMP7, MAP3K7IP1, and HIPK2 (Luo et al., 2013), are associated with chicken HVP.

In recent years, proteomics technology has provided new perspectives for elucidating complex phenotypes. Data-independent acquisition (DIA) proteomics offers extensive protein coverage, high reproducibility, and accuracy (Li et al., 2021). The quantitative values of many proteins obtained through DIA can rival those of targeted methods (He et al., 2019). Proteomics is commonly used to study the pathogenesis of various diseases (Song et al., 2023b) and is increasingly applied to research various color traits in chickens, including earlobe color (Li et al., 2024), feather color (Leskinen et al., 2012; Wang et al., 2019), and white stripes(Kong et al., 2024). This study selected bearded chickens at 40 and 120 d of age, utilizing DIA proteomics sequencing to obtain protein profiles of different grades of peritoneum, aiming to identify key pathways and candidate proteins related to HVP formation, and to explore the effect of the presence of HVP on broilers.

Materials and methods

Ethics statement

The experimental animals were provided by Longmen Xingtai Modern Agriculture Co., Ltd. (Huizhou City, Guangdong Province, China). The animal experiments were reviewed and approved by the Animal Ethics Committee of South China Agricultural University (approval number: SCAU#0106; 25 November 2018).

Experimental animals

Sixty female chicks at 40 d of age and 30 female chicks at 120 d of age from the same batch were used, and the experimental animals were fed according to standard requirements and provided with standard drinking water. The room temperature was maintained at approximately 26°C, and the humidity is 50 %−60 %. After anesthesia, the chickens were sacrificed by cutting off the jugular vein, bloodletting for 3-5 min, and then dissected. The peritoneum is removed, and samples are classified based on the size of the melanotic spots on the peritoneum into completely HVP (B), faded (F), and normal (N) peritoneum (Fig. 1). Each group has three replicates, and they are named according to age as 40B, 40F, 40 N, 120B, 120F, and 120 N for DIA protein sequencing. Additionally, peritoneal samples are collected and fixed in 4 % paraformaldehyde for paraffin sectioning. In addition, three 120 d of age female red crown black skin chicken were selected for the same treatment, and peritoneal samples were collected for DIA proteomics analysis (the group is named RCBSC.).

Fig. 1.

Fig. 1

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Flowchart.

Hematoxylin and eosin and fontana-masson staining

After cutting the peritoneum to a suitable size, it was stored in a formalin solution. Then, according to the experimental procedure, the peritoneal samples were fixed in 4 % paraformaldehyde solution for H&E and Fontana-Masson staining. Tissue pathological observations were made using an optical microscope.

Determination of peritoneal melanin content

The peritoneum was weighed and mixed with PBS at a ratio of 1: 9 to prepare the test sample. After being cut into small pieces with ophthalmic scissors, the samples were homogenized in a homogenizer. The mixture was then centrifuged at 3000 rpm for 10 min, and the supernatant was collected. The procedure was carried out according to the instructions provided in the Chicken MLELISA Kit (YuDuo, Shanghai), and the OD value was measured at a wavelength of 450 nm.

On-machine protein mass spectrometry

Protein extraction and peptide digestion. For each sample, an appropriate amount of SDT lysis buffer (4 % SDS, 100 mM Tris-HCl, pH 7.6) was added to extract the protein, and protein quantification was carried out using the BCA method. A portion of 15 µg of protein from each sample was mixed with a suitable amount of 5X loading buffer and heated in a boiling water bath for 5 min. SDS-PAGE electrophoresis was performed using a 4 %−20 % pre-cast gradient gel at a constant voltage of 180 V for 45 min, followed by staining with Coomassie Brilliant Blue R-250.

All samples were digested using the filter aided proteome preparation method with trypsin. The peptides from the digested samples were desalted using a C18 cartridge, and after lyophilization, the peptides were reconstituted in 40 μL of 0.1 % formic acid solution. The peptide concentration was determined by measuring OD280. An appropriate amount of iRT standard peptides was added to each sample's digested peptides, and DIA mass spectrometric detection was performed using the Astral high-resolution mass spectrometer.

DIA sample on-machine. DIA analysis was conducted using the Vanquish Neo system (Thermo Fisher) for chromatographic separation at a nanoscale flow rate. The samples separated by nanoscale high-performance liquid chromatography were analyzed using the Astral high-resolution mass spectrometer (Thermo Scientific) for DIA mass spectrometry analysis. The detection mode was positive ions, with a precursor ion scan range of 380-980 m/z. The first stage mass spectrometry resolution was 240,000 at 200 m/z, with a Normalized AGC Target of 500 %, and Maximum IT of 5 ms. The MS2 utilized the DIA data acquisition mode, configured with 299 scanning windows, an Isolation Window of 2 m/z, HCD Collision Energy of 25 eV, a Normalized AGC Target of 500 %, and Maximum IT of 3 ms.

DIA statistical analysis. The DIA data were analyzed for database searching and protein quantification using DIA-NN software (v. 1.8.1). The database searching process employed the Library-free method, utilizing the uniprot_fanya_iRT_20230130.fasta database, which contains 28,714 sequences. In the database searching settings, deep learning-based parameters were enabled to predict the spectral library and Match Between Runs (MBR) was selected. This allowed for the generation and re-analysis of the spectral library from the DIA data, facilitating protein quantification, which was performed based on the MaxLFQ algorithm. The software parameter settings are as follows. The enzyme is trypsin, the max miss cleavage site is 1, the fixed modification is Carbamidomethyl (C), and the dynamic modifications are set to Oxidation (M) and Acetyl (Protein N-term). The peptide sequences and proteins identified through database searching must meet the established filtering parameter of FDR (false discovery rate) < 1 %. The differential analysis employed the t-test method, with the filtering criteria for differential abundance proteins (DAPs) set at FC > 1.5 or < 0.66 and a P-value < 0.05. The BH method is used to correct the P-value.

Protein-protein interaction network

DAPs were input into the STRING database (http://string-db.org/) to find interactions between proteins using a minimum interaction score threshold of 0.4 (middle confidence). The TSV format file of Protein-Protein Interaction Network (PPI) was downloaded and imported into Cytoscape software to construct the PPI network.

GO and KEGG annotation and enrichment

The DAPs were functionally annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg) databases and Gene Ontology (GO, http://www.geneontology.org). In the GO enrichment analysis, only upregulated DAPs were selected. We performed enrichment analysis using the R package clusterProfiler (v. 4.12.6). Terms with a P-value < 0.05 were considered significantly enriched, and Top10 was selected for mapping.

WGCNA analysis of HVP protein abundance profile

Weighted gene co-expression network analysis (WGCNA) analysis was performed using R package WGCNA (v.1.73), we removed low-abundance proteins and those with stable expression. Based on a scale-free network topology, the soft threshold was automatically calculated using functions. Proteins with similar expression were divided into the same module. We selected the module with the highest correlation to group B based on correlation and significance for KEGG and GO enrichment analysis.

Soft clustering of HVP progression-related proteins

To study the overall protein expression patterns, a soft clustering method known as fuzzy c-means clustering was implemented using the R package Mfuzz (v. 2.48.0). In brief, the average protein expression values at each stage (N, F, and B) were first calculated and then fed into the fuzzy c-means algorithm, resulting in the generation of several protein clusters, each with distinct expression patterns.

Transcription factor analysis

The prediction of transcription factor families was conducted using the public database AnimalTFDB4.

Analysis of structure domain

The InterProScan software was used to perform functional characterization of the sequence by running the scanning algorithm from the InterPro database in an integrated manner to obtain the domain annotation information of DAPs sequence in the Pfam database.

GSEA analysis

For the gene set enrichment analysis (GSEA) analysis of DAPs, the R packages msigdbr (v. 7.5.1) and fgsea (v. 1.30.0) were selected. The Symbol genes of the proteins in the DAPs were converted in advance to the homologous Entrezid genes in the human genome. Subsequently, GSEA analysis was performed using the human database ‘org.Hs.eg.db’. The KEGG and GO enrichment analyses were conducted using the built-in functions gseKEGG and gseGO, respectively.

Western blot analysis

After the peritoneal samples were disrupted, they were lysed in RIPA lysis buffer, and the protein concentration was measured using the BCA method (Beyotime, China). After denaturation, proteins were separated by SDS-PAGE and transferred to a PVDF membrane, which was blocked with a rapid blocking solution (Beyotime, China) for 15 min. The membrane was incubated overnight with primary antibodies: Rabbit Anti-DHRS3 antibody (Affinity, Australia) and Rabbit Anti-VCAN antibody (A19655) (ABclonal, China), followed by incubation with secondary antibodies for 1 h. The detection solution was prepared according to the instructions of ultra-sensitive ECL chemiluminescent reagent kit (Beyotime, China), and the results were visualized using an ECL chemiluminescence imaging system, with analysis performed using ImageJ software.

Results

Peritoneal characteristics of different phenotypes of chickens

The melanin content in the black peritoneum, faded peritoneum, and normal peritoneum of chickens at 40 d of age were 37.71, 25.32, and 28.61 mg/mL, respectively (Fig. 2A). At 120 d of age, the melanin content in the black peritoneum, faded peritoneum, and normal peritoneum of chickens were 35.78, 23.54, and 28.47 mg/mL, respectively (Fig. 2B). There was a significant difference between the black peritoneum and the normal peritoneum of chickens at both 40 and 120 d of age (P-value < 0.05). Similarly, there is a significant difference between the black peritoneum and the faded peritoneum of chickens, but no significant difference between the faded peritoneum and the normal peritoneum of chickens.

Fig. 2.

Fig. 2

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Comparison of characteristics between black peritoneum and normal peritoneum. (A) Measurement of melanin content in the peritoneum at 40 d of age. (B) Measurement of melanin content in the peritoneum at 120 d of age. (C) H&E and Fontana-Masson staining of the peritoneum, scale bars: 50 μm.

To gain a more detailed understanding of the morphological changes and melanin deposition of the black peritoneum, we performed H&E and Fontana-Masson staining followed by optical microscopy observation (Fig. 2C). Results showed that the melanin in the black peritoneum exhibited a clustered distribution rather than being evenly dispersed throughout the peritoneum. Instead, it existed in the form of clumps, demonstrating significant heterogeneity. In areas with melanin accumulation, the number of cell nuclei was also significantly increased. Therefore, the formation of the black peritoneum is attributed to the local increase in melanocytes, leading to a substantial production of melanin.

Differences in proteins between B and N peritoneum

Based on DIA proteomics technology, this study identified a total of 9,375 high-confidence proteins, covering 159,879 specific peptides. Partial least squares discriminant analysis showed a clear clustering separation in the protein profiles of HVP samples at 40 and 120 d of age based solely on the proteomic patterns (Fig. 3A–B). Based on the melanin detection results, we compared the black peritoneum at 40 and 120 d of age with the normal peritoneum of the same age (designated as 40B-N and 120B-N, respectively). In the 40B-N group, we identified 178 upregulated DAPs and 41 downregulated DAPs. In the 120B-N group, we identified 174 upregulated DAPs and 72 downregulated DAPs (Fig. 3C–D, Supplementary Table 1). According to the screening results, the B group and N group exhibited different protein abundance patterns (Fig. 3E–F). There were only two co-expressed proteins between the two groups of DAPs: DHRS3 and DACT1 (Fig. 3G). Among the DAPs at 40 d, proteins related to melanin synthesis, such as DCT and TYRP1, were significantly enriched. However, no significant differences were observed between the two groups at 120 d of age.

Fig. 3.

Fig. 3

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Overview of proteomics. (A) PLS-DA of different grades of peritoneum at 40 d of age. (B) PLS-DA of different grades of peritoneum at 120 d of age. (C) Volcano plots of 40B-N. (D) Volcano plots of 120B-N. (E) Hierarchical clustering of DAPs in the 40B-N. (F) Hierarchical clustering of DAPs in the 120B-N. (G) Venn diagram of DAPs between 40B-N and 120B-N, red: up-regulated proteins, green: down-regulated proteins, gray: protein abundance trend change.

The biological function and signaling pathway enrichment analyses were conducted for the DAPs at two different ages. In the 40B-N group, from the KEGG signal pathway analysis, the upregulated DAPs are mainly enriched in the ribosome, as well as in tyrosine metabolism and melanogenesis, while the downregulated DAPs are primarily enriched in nitrogen metabolism, cell adhesion molecules, p53 signaling pathway, and PPAR signaling pathway (Fig. 4A). At the Level 2 hierarchy, translation and cell growth and death show significant enrichment (Fig. 4B). GO analysis shows that 219 DAPs are primarily enriched in the MF aspects of structural constituent of ribosome and structural molecule activity; CC includes cytosolic large ribosomal subunit, large ribosomal subunit, and ribosome; BP aspects are positive regulation of excitatory postsynaptic potential, translation, and peptide biosynthesis process (Fig. 4C–E). In the PPI network constructed using the string website, the largest cluster is related to the function of ribosomes (Fig. 4F).

Fig. 4.

Fig. 4

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GO and KEGG analysis of 40B-N. (A) KEGG enrichment analysis, red indicates up-regulation, and blue indicates down-regulation. (B) Bar chart of KEGG pathway assignments. (C-E) GO enrichment analysis: (C) CC, (D) MF, (E) BP. (F) PPI network.

In the 120B-N group, from the KEGG signal pathway analysis, the upregulated DAPs are mainly enriched in the spliceosome, steroid biosynthesis, and Wnt signaling pathway, while the downregulated DAPs are primarily enriched in the biosynthesis of unsaturated fatty acids, nucleocytoplasmic transport, and PPAR signaling pathway (Fig. 5A). At the Level 2 hierarchy, transport and catabolism, cell growth and death, and signal transduction show significant enrichment (Fig. 5B). GO analysis shows that 246 DAPs are primarily enriched in the MF aspects of sequence-specific DNA binding, DNA-binding transcription factor activity, RNA polymerase II-specific, and RNA polymerase Ⅱ transcription regulatory region sequence-specific DNA binding related to transcriptional functions; CC aspects include extracellular space, collagen type I trimer, and CAF-1 complex; and BP aspects are response to ischemia, negative regulation of vascular-associated smooth muscle cell differentiation, and the Toll-like receptor 7 signaling pathway (Fig. 5C–E). In the PPI network, the largest cluster is related to the processing of mRNA synthesis (Fig. 5F).

Fig. 5.

Fig. 5

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GO and KEGG analysis of 120B-N. (A) KEGG enrichment analysis. (B) Bar chart of KEGG pathway assignments. (C-E) GO enrichment analysis: (C) CC, (D) MF, (E) BP. (F) PPI network.

WGCNA of HVP tissue protein abundance patterns

WGCNA is a systems biology approach used to identify co-expressed protein networks within a large set of proteins (Fig. 6A). Eighteen different modules were obtained using a protein dendrogram colored according to correlations between protein abundance levels. The darkgreen module had the highest correlation with HVP at 40d (r = 0.81, P-value = 0.001), and the darkred module had the highest correlation with HVP at 120d (r = 0.6, P-value = 0.04) (Fig. 6B–C, Supplementary Table 2).

Fig. 6.

Fig. 6

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Weighted gene coexpression network analysis. (A) Hierarchical clustering tree of isolated modules displayed in various colors, with the gray module representing a group of genes that cannot be included in other modules. (B) Module-trait relationships within the network, where the upper number in each row shows the correlation with the corresponding phenotypic traits, while the lower number indicates the related P-values. (C) Expression levels of module eigengenes in the dark green and dark red modules across various traits. (D) GO analysis of the proteins associated with the dark green and dark red modules. (E) KEGG enrichment analysis of the proteins related to the dark green and dark red modules.

We performed KEGG and GO analysis for these two modules. In the darkgreen module, KEGG pathways related to the nicotinate and nicotinamide metabolism, nitrogen metabolism, and GnRH signaling pathway were enriched. GO terms related to transcription regulator complex, melanosome, and pigment granule were enriched. In the darkred module, KEGG pathways related to the glycine, serine and threonine metabolism, extracellular matrix (ECM)-receptor interaction, and ATP-dependent chromatin remodeling were enriched. GO terms related to histone deacetylase complex, histone binding, and ATPase complex were enriched (Fig. 6D–E). The KEGG enrichment and GO analysis of the modules screened by WGCNA had multiple coincidences with the results of DAPs. The presence of niacin and nicotinamide metabolism, along with the GnRH signaling pathway, at 40 d of age, suggests that steroid hormones are involved in HVP. Among the DAPs, we identified HSD17B12, which is responsible for synthesizing estradiol and also plays a role in lipid metabolism in the body. At 120 d of age, HVP chickens facilitate chromatin remodeling and histone deacetylation through the extensive synthesis of ATP, thereby enhancing transcriptional efficiency.

Soft clustering of HVP tissue protein abundance patterns

We used fuzzy algorithms to assess the longitudinal evolution of the average abundance of proteins related to three degrees of peritoneal melanin deposition. The clustering results for the two different ages are shown in Fig. 7A–B, each consisting of six protein abundance patterns. The abundance patterns are represented by colored trend lines and are characterized by the fluctuations of all proteins in N, F, and B.

Fig. 7.

Fig. 7

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Mfuzz analysis reveals the different expression patterns of proteins in HVP. (A) Protein expression pattern at 40 d of age. (B) Protein expression pattern at 120 d of age. (C) Heatmap and KEGG enrich of six protein clusters at 40 d of age. (D) Heatmap and KEGG enrich of six protein clusters at 120 d of age.

At 40 d, Group B showed significant changes in the average protein abundance in clusters 3 and 5, with an increase in cluster 3 and a decrease in cluster 5. In C3, the KEGG results were related to cell adhesion and melanogenesis, suggesting that C3 may be associated with the migration and function of melanocytes. At 120 d, Group B exhibited notable changes in average protein abundance in clusters 1 and 2, with an increase in cluster 1 and a decrease in cluster 2. We identified several proteins from clusters with similar average abundance trends across the two ages, with SCG2 showing increased average abundance and LCAT showing decreased average abundance. GO enrichment analysis of the proteins for each cluster revealed that both cluster C5 at 40 d and cluster C2 at 120d were commonly enriched in the PPAR signaling pathway and fat-related pathways (Fig. 7C–D). These pathways were also co-expressed in the KEGG enrichment analysis of the two groups of DAPs. These results suggest that the presence of HVP inhibits fat deposition in broilers.

Difference in protein between 120B and 40B

By comparing the protein profiles of 120B and 40B, we identified 273 DAPs, with 89 being upregulated and 184 downregulated (Fig. 8A–B, Supplementary Table 1). KEGG enrichment analysis of these 273 DAPs revealed that pathways involved in the synthesis of chondroitin sulfate/dermatan sulfate, the TCA cycle, ferroptosis, and mitochondrial autophagy were upregulated; conversely, pathways related to glycosaminoglycan degradation, ABC transporters, and other polysaccharide degradation were downregulated (Fig. 8C). The 273 DAPs were primarily enriched in BP related to antigen processing and presentation, as well as defense responses to symbionts and viruses; in CC, they were enriched in major histocompatibility complex (MHC) class Ⅱ protein complexes, extracellular space, and the extracellular side of the plasma membrane; in MF, they were enriched for transmembrane signaling receptor activity, ABC peptide transporter activity, and peptide transmembrane transporter activity (Fig. 8D–F).

Fig. 8.

Fig. 8

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Comparison of HVP development. (A) Hierarchical clustering of DAPs in the 120B-40B. (B) Volcano plots of 120-40B. (C) KEGG enrichment analysis. (D-F) GO enrichment analysis: (D) BP, (E) CC, (F) MF.

The increased synthesis and decreased degradation of glycosaminoglycans, along with the upregulation of tight junctions, ECM-receptor interactions, and focal adhesion pathways, indicate changes in the ECM matrix components in 120B. Compared to 40B, the abundance of glycosaminoglycans increased in 120B, and the abundance of the relevant ECM protein VCAN returned to normal levels. We believe that this increased synthesis may compensate for the earlier reduction in glycosaminoglycan levels during the HVP formation process. It may also suggest that the formation of HVP occurs in two stages: in the early stage, fibroblasts mediate melanocyte colonization by altering ECM matrix components, while in the later stage, changes in ECM components inhibit further spread. Moreover, we observed a significant downregulation of functions related to the major histocompatibility complex MHC in GO, including both class I and Ⅱ types. Although the presence of HVP is not a result of immune dysfunction, the accumulation of peritoneal pigment gradually reduces the immune response capability of broiler chickens.

Domain analysis and transcription factor of DAPs

In the 40B-N group, DAPs were significantly enriched in tyrosinase domains, globin domains, α/β hydrolase fold domains, and silk protein domains (Fig. 9A); in the 120B-N group, DAPs were significantly enriched in keratin type Ⅱ domains, silk protein domains, serine proteinase domains, and gelatin domains (Fig. 9B). The globin domains, keratin domains, and serine proteinase domains are related to the ECM matrix, suggesting that the formation of HVP is associated with the components of the ECM matrix.

Fig. 9.

Fig. 9

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Protein domain analysis and transcription factor prediction. (A) Barplot of domain analysis of DAPs in 40B-N. (B) Barplot of domain analysis of DAPs in 120B-N. (C) Barplot of transcription factor annotation for 40B-N. (D) Barplot of transcription factor annotation for 120B-N.

Through transcription factors (TFs) analysis, the DAPs from groups 40B-N and 120B-N were enriched in families such as ZBTB, zf-C2H2, and TF-bZIP. The commonly enriched TFs included ZBTB37 and ZFP91 (Fig. 9C–D). The enriched TFs families are all associated with zinc finger domains. Therefore, we believe that TFs containing zinc finger domains play an important role in the formation of HVP.

Difference in protein between 120B and RCBSC

WMC is a local chicken breed in Guangdong Province, China, similar to Silky fowl (SF), and also exhibits systemic pigmentation, but with an average blackness lower than that of SF. We used RCBSC as a control to explore the protein differences between local pigmentation in 120B and systemic pigmentation in RCBSC. The 120B group showed a different protein expression pattern compared to RCBSC (Fig. 10A), with a total of 2,744 DAPs identified (2,571 upregulated and 173 downregulated) (Fig. 10B, Supplementary Table 1). GSEA analysis of these DAPs revealed activation of KEGG pathways such as protein digestion and absorption, serotonergic synapse, and protein processing in the endoplasmic reticulum (Fig. 10C). The activated entries in GO were related to ECM and extracellular space, while ribosome-related entries were suppressed (Fig. 10D). The pathways of protein digestion and absorption, ECM-receptor interaction, and cytoskeleton in muscle cells formed interactions (Fig. 10E–F).

Fig. 10.

Fig. 10

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The protein difference between 120B and RCBSC. (A) Hierarchical clustering of DAPs in the 120B-RCBSC. (B) Volcano plots of 120B-RCBSC. (C-D) KEGG and GO enrichment in GSEA analysis. (C) KEGG, (D) GO. (E) GSEA rank plot. (F) KEGG entries interaction network.

There were significant differences in extracellular matrix components between 120B and RCBSC, with upregulated abundances of collagen COL4A4, COL2A1, COL16A1, COL12A1, COL6A3, COL1A2, COL6A1, COL1A1, COL5A1, COL3A1, COL6A2 and COL8A2, core proteoglycans VCAN, DCN and BGN, indicating that the extracellular matrix in the abdominal area of the 120B group is denser. The upregulation of integrin family proteins ITGB5, ITGAV, ITGBL1, ITGB9, ITGA1, ITGB1, ITGA3, ITGB6 and ITGB4, fibronectin FN1, and myosins MYH9 and MYH10, are related to the migration of NCCs lineage cells, indicating that 120B peritoneum has a more suitable microenvironment for the migration of NCCs lineage cells. Although HVP is different from fibromelanosis, we speculate that with the deposition of melanin, the activation of TGF-β and PI3K-Akt signaling pathways, and the increase of collagen, the peritoneum may initiate the fibrosis process.

Discussion

HVP significantly reduces the quality of chicken carcasses, affecting their economic value. Unlike peritoneal melanosis, the formation of HVP is not due to disease or tumors but originates from regulatory processes during development. Its formation involves a complex network of cellular interactions and molecular regulation. We screened two key proteins, DACT1 and DHRS3, both of which directed to the function of NCCs, with NCCs originating from the closed neural tube, representing a migratory population of multipotent stem cells. This transient population consists of four subgroups designated along the body axis from the beak to the tail as cranial, vagal, trunk, and sacral(Jacobs-Li et al., 2023). Neural crest cells can produce a variety of cell types, including melanocytes, most peripheral neurons and glial cells (Vandamme and Berx, 2019). In newly hatched birds, NCCs exhibit two migration patterns: early neuronal NCCs migrate ventromedially after entering the ectoderm, while later NCCs fated to become melanocytes migrate dorsolaterally, invading and colonizing the epidermis (Schwarz et al., 2009). The delamination of NCCs is designated by the sequential and coordinated activity of at least five different signaling pathways: bone morphogenetic protein, Wnt, fibroblast growth factor, retinoic acid (RA), and Notch (Schwarz et al., 2009; Betancur et al., 2010; Mayor and Theveneau, 2013; Rabadan et al., 2016).

DACT1 is a scaffold protein and one of the essential factors for the delamination of NCCs, the delamination of NCCs refers to the critical step during embryonic development where they detach from the outer edge of the neural plate or neural tube, forming an independent population of cells. This process marks the separation of NCCs from the primitive ectodermal tissue and initiates their migration and differentiation. DACT1 can inhibit the classic Wnt signaling pathway by specifically suppressing the transcriptional activity of β-catenin and regulating subcellular localization, thereby inhibiting the generation of melanocytes (Szemes et al., 2019). However, it can also activate this pathway, depending on the phosphorylation state of DACT1 (Contriciani et al., 2021). The paralog DACT2, when overexpressed in chicken embryos, enhances NCC migration and increases the number of melanocytes, while overexpression of DACT1 in Xenopus laevis embryos similarly enhances the migratory capacity of NCCs (Rabadan et al., 2016). DACT1 may affect the delamination of NCCs by regulating the Wnt signaling pathway, promote the differentiation of melanocytes, and promote the migration of NCCs.

The efficiency of melanocyte migration is closely related to the precise coordination of ECM adhesion mediated by integrins (Haage et al., 2020), where proteoglycans such as chondroitin sulfate are essential elements for NCC migration (Gouignard et al., 2016). VCAN was identified in 40B-N DAPs, it is a chondroitin sulfate proteoglycan, acts as a barrier molecule hindering the migration and invasion of NCCs (Szabo et al., 2016). In SF, the abnormal migration patterns of NCCs may arise from the absence of barrier molecules. The reduced abundance of sulfated proteoglycans like VCAN weakens the barrier function of the peritoneum, allowing more melanocytes to migrate and colonize the peritoneum. In the 120B-RCBSC comparison, we identified a significant number of integrin family proteins, which were not observed in the other comparison groups. We hypothesize that the upregulation of integrin abundance may guide the aggregation of melanocytes, but it is not the primary factor in the formation of HVP. Compared with 120B, the further compromised barrier function in RCBSC may promote the extensive invasion and colonization of melanocytes, which might explain their widespread distribution in tissues.

HSD17B12 was found in both the darkgreen module and 40B-N, which is responsible for the synthesis of estradiol and participates in lipid metabolism in vivo. Estradiol, as a sex hormone, can extend the laying period and increase egg production in hens through repeated elevation (Sun et al., 2021; Shi et al., 2024). In vitro experiments have shown that estradiol promotes the proliferation of melanocytes and stimulates pigmentation (Wiedemann et al., 2009). Differences in the genes HSD17B1 and the metabolite estradiol were found in the metabolism and lipid profiles of Silkie chickens compared to other chickens of ordinary quality(Yang et al., 2024). This suggests that the differential synthesis of estradiol might lead to similar melanin synthesis and deposition (Tian et al., 2021). Therefore, we believe that estradiol may also be a differential metabolite of HVP.

In our preliminary investigations, although HVP occurs in most broiler chicken breeds, its frequency is higher in Huizhou bearded chickens compared to other breeds. The bearded chicken is known for its elongated feathers located on both sides of the face and below the beak, which is genetically based on the specific expression of HOX family genes and differences in RA levels (Zheng et al., 2023). This aligns with our findings that variations in RA levels also induce the emergence of HVP, suggesting that the bearded trait could aid in the selection of HVP traits. The high abundance of DHSR3 may stem from the body's progressive regulation of RA levels, prevent excessive RA generation (Cerignoli et al., 2002; Adams et al., 2014). Previous researchers have identified CYP2D6 as a gene highly associated with HVP. CYP2D6 is a major drug-metabolizing enzyme and, as a member of the cytochrome P450 family, is involved in the metabolism of fatty acids, steroids, and vitamin A derivatives (Li et al., 2017; Ning et al., 2019). This is consistent with our findings, as we believe that HVP chickens are in an active phase of metabolizing these substances. Endogenous RA significantly influences the delamination of NCCs (Dupin and Le Douarin, 1995; Rekler et al., 2024) and has a bimodal effect on melanocyte differentiation. In the early stages, it promotes the differentiation of melanocyte precursors by inducing tyrosinase, while in the later stages, apoptosis removes terminally differentiated melanocytes (Watabe et al., 2002; Inoue et al., 2012; Johns et al., 2025). It also exhibits a bimodal effect on melanin deposition (Inoue et al., 2012). In vertebrates, higher levels of RA can significantly enhance the expression of tyrosinase genes and melanin deposition (Jin et al., 2022; Yu et al., 2024). We believe that excessive RA in vivo induces the differentiation of melanocytes and promotes the deposition of melanin together with estradiol.

The widespread melanin deposition in Silkie chickens has a significant impact on their immune capacity. In the early stages (1 d to 6 w), it suppresses the development of immune organs, but in later stages, it delays the degeneration of these organs (Han et al., 2015). In this study, we found that as the duration of pigment deposition increased, the immune response capability of HVP chickens decreased. The downregulation of MHC-related proteins reduced the chickens' resistance to avian diseases such as MDV, ALV, and NDV.

In various studies comparing black phenotypes to other color phenotypes, enrichment analyses of DAPs or differentially expressed genes have been associated with ribosomal function and protein synthesis (Fan et al., 2013; Song et al., 2023a; Tan et al., 2024). This suggests that active protein synthesis might be a common key characteristic of excessive melanin deposition. Additionally, transcriptional enhancement may be related to the reduction of phototoxicity caused by excessive melanin deposition (Zamudio Diaz et al., 2024), as DNA damage can shut down global transcription to prevent transcriptional mutagenesis and initiate repair signaling (Fu et al., 2022). At 40 d, pathways related to energy metabolism, such as glycolysis/gluconeogenesis and fatty acid elongation, were enriched. The synthesis of melanin requires a significant supply of energy, and the presence of fatty acids enhances mitochondrial respiration during the pigmentation process (Sultan et al., 2022). Additionally, the substantial synthesis of proteins also demands energy supply. It indicated that the formation and maintenance of HVP required a lot of energy, resulting in weight loss of HVP chickens.

Conclusions

This study elucidates the molecular mechanisms underlying the occurrence of HVP. The melanocytes in the peritoneum originate from the directed differentiation of NCCs induced by RA and stimulated by DACT1. The reduction in the abundance of chondroitin sulfate, represented by VCAN, weakens the peritoneal barrier function. The hormonal microenvironment created by estradiol, along with the presence of RA, promotes melanin synthesis in melanocytes, leading to melanin deposition. Additionally, the need to synthesize large amounts of melanin to form the HVP phenotype requires substantial energy expenditure, resulting in reduced body weight in broilers. Furthermore, the continuous deposition of melanin is associated with a decline in the immune response capacity of the broilers.

DATA AVAILABILITY STATEMENT

The original sequencing data of peritoneal proteomics of bearded chicken has been uploaded to the OMIX database, Bioproject number: OMIX008484, and can be accessed directly at https://ngdc.cncb.ac.cn/omix/preview/NL7g9JuW.

Author contributions

Zhengyang Chen conceived and performed the experiments, analyzed the data, and wrote the manuscript. Changbin Zhao conceived the experiments. Rong Fu, Chengyue Yuan and Ke Zhang revised the manuscript. Xiquan Zhang conceived this study, revised and approved the final manuscript. All authors contributed to the article and approved the submitted version.

Funding

This study was supported by the ''China Agriculture Research System (Grant No. CARS-41) '', and National Key Research and Development Program of China(2021YFD1300102).

Declaration of competing interest

We confirm that this work is original and has not been published elsewhere nor is it currently under consideration for publication elsewhere.

Footnotes

☆

Appropriate scientific section for the paper: Genetics and Molecular Biology.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.psj.2025.105114.

Appendix. Supplementary materials

mmc1.xlsx (373.4KB, xlsx)
mmc2.xlsx (19.2KB, xlsx)
mmc3.zip (604KB, zip)

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Associated Data

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Supplementary Materials

mmc1.xlsx (373.4KB, xlsx)
mmc2.xlsx (19.2KB, xlsx)
mmc3.zip (604KB, zip)

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