Transcriptomic and epigenomic insights into pectoral muscle fiber formation at the late embryonic development in pure chicken lines

Abstract

Long-term intensive genetic selection has led to significant differences between broiler and layer chickens, which are evident during the embryonic period. Despite this, there is a paucity of research on the genetic regulation of the initial formation of muscle fiber morphology in chick embryos. Embryonic d 17 (E17) is the key time point for myoblast fusion completion and muscle fiber morphology formation in chickens. This study aimed to explore the genetic regulatory mechanisms underlying the early muscle fiber morphology establishment in broiler chickens of Cornish (CC) and White Plymouth Rock (RR) and layer chickens of White Leghorn (WW) at E17 using the transcriptomic and chromatin accessibility sequencing of pectoral major muscles. The results showed that broiler chickens exhibited significant higher embryo weight and pectoral major muscle weight at E17 compared to layer chickens (P = 0.000). A total of 1,278, 1,248, and 892 differentially expressed genes (DEGs) of RNA-seq data were identified between CC vs. WW, RR vs. WW, and CC vs. RR, separately. All DEGs were combined for cluster analysis and they were divided into 6 clusters, including cluster 1 with higher expression in broilers and cluster 6 with higher expression in layers. DEGs in cluster 1 were enriched in terms related to macrophage activation (P = 0.002) and defense response to bacteria (P = 0.002), while DEGs in cluster 6 showed enrichment in protein-DNA complex (P = 0.003) and monooxygenase activity (P = 0.000). ATAC-seq data analysis identified a total of 38,603 peaks, with 13,051 peaks for CC, 18,780 peaks for RR, and 6,772 peaks for WW. Integrative analysis of transcriptomic and chromatin accessibility data revealed GOLM1, ISLR2, and TOPAZ1 were commonly upregulated genes in CC and RR. Furthermore, screening of all upregulated DEGs in cluster 1 from CC and RR identified GOLM1, ISLR2, and HNMT genes associated with neuroimmune functions and MYOM3 linked to muscle morphology development, showing significantly elevated expression in broiler chickens compared to layer chickens. These findings suggest active neural system connectivity during the initial formation of muscle fiber morphology in embryonic period, highlighting the early interaction between muscle fiber formation morphology and the nervous system. This study provides novel insights into late chick embryo development and lays a deeper foundation for further research.

Keywords: ATAC-seq; RNA-seq; chicken; embryonic period; muscle development.

Figure 1

Embryonic growth patterns of broiler and layer chickens. (A) The body weight (BW) of individuals with Cornish (CC), White Plymouth Rock (RR) and White Leghorn (WW) at the embryonic d 17 (E17). (B) The pectoral major muscle weight (PMW) of individuals with CC, RR, and WW at E17. (C) Pectoral major muscles were collected at D21 and D42 for RNA-seq and ATAC-seq analysis. ^⁎⁎ and * indicate P-value less than 0.01 and 0.05, respectively.

Figure 2

Transcriptomic analysis of pectoral major muscle at E17. (A) PCA plot of RNA-seq data of CC, RR. and WW groups at E17. Each point represents a muscle sample. (B) Volcano plot of differential expressed genes (DEGs) obtained between CC and WW. (C) Volcano plot of differential expressed genes (DEGs) obtained between RR and WW. (D) Different expression patterns among the 3 experimental groups of all DEGs. (E) Heatmaps of intergroup expression of DEGs contained in cluster 1 and cluster 6. (F) GO enrichment results of DEGs of cluster 1 and cluster 6.

Figure 3

Analysis of chromatin accessibility from the pectoral muscle. (A) PCA plot of ATAC-seq data of CC, RR and WW groups at E17. Each point represents a muscle sample. (B) Overlap peaks between 2 groups. (C) The functional region distributions of ATAC-seq peak dataset. (D) The predicted motifs for the upregulated peaks of CC between CC and WW. (E) The predicted motifs for the upregulated peaks of RR between RR and WW.

Figure 4

Integration of RNA-seq and ATAC-seq for candidate gene identification. (A)Venn diagrams showed the overlap of DEGs and the associated genes of different peaks between CC and WW. (B) Venn diagrams showed the overlap of DEGs and the associated genes of different peaks between RR and WW. (C) The relationship between gene expression and open chromatin regions. (D) UpSet plot of 2 classes of the upregulated DEGs. (E) GO enrichment analysis of DEGs filtered from cluster 1.

Figure 5

Expression analysis of candidate genes. Differential expression analysis of ISLR2, MYOM3, GOLM1, HNMT, GAA, MYLK4, BATF3, and FGF19 genes between broiler chickens (CC and RR) compared to layer chicken (WW).