Transcriptome and co-expression network analysis of the chick blastoderm during egg storage prioritize CTGF as a...

Embryonic diapause is a reversible developmental arrest that enables embryos to survive unfavorable conditions. In poultry production, fertilized eggs are routinely stored at sub-incubation temperatures before setting, during which the chick blastoderm remains at an early stage and developmental progression is effectively restrained. However, the transcriptional basis underlying entry into and exit from this storage-induced dormant state remains poorly understood. Here, we performed RNA-seq on chick blastoderms collected at 3 stages, including freshly laid, cold-stored, and 4 h after rewarming. Differential expression analyses identified flip differentially expressed genes (flip DEGs) that changed in opposite directions during entry into and exit from storage, suggesting a reversible transcriptional program. Weighted gene co-expression network analysis (WGCNA) identified the module most strongly associated with the dormant state. By intersecting module genes with flip DEGs and applying an integrated ranking framework, we prioritized connective tissue growth factor (CTGF) as a candidate associated with the dormant state. CTGF overexpression in chick embryonic fibroblasts reduced proliferation, increased the G0/G1 fraction, and decreased apoptosis, consistent with dormancy-like cellular features. RNA-seq of CTGF-overexpressing fibroblasts, followed by Hallmark gene set enrichment analysis (GSEA), showed enhanced unfolded protein response and protein secretion programs together with reduced inflammatory, interferon, and p53/apoptosis-related signaling. These CTGF-associated transcriptional changes partially overlapped with storage-associated transcriptional features in blastoderms. Collectively, our findings characterize a reversible transcriptional program associated with storage-induced dormant state and nominate CTGF-associated remodeling and downstream transcriptomic features as candidate molecular features of embryo adaptation to egg storage.