摘要Background Four-chambered stomach including the forestomachs(rumen,reticulum,and omasum)and aboma-sum allows ruminants convert plant fiber into high-quality animal products.The early development of this four-cham-bered stomach is crucial for the health and well-being of young ruminants,especially the immune development.However,the dynamics of immune development are poorly understood.Results We investigated the early gene expression patterns across the four-chambered stomach in Hu sheep,at 5,10,15,and 25 days of age.We found that forestomachs share similar gene expression patterns,all four stomachs underwent widespread activation of both innate and adaptive immune responses from d 5 to 25,whereas the meta-bolic function were significantly downregulated with age.We constructed a cell landscape of the four-chambered stomach using single-cell sequencing.Integrating transcriptomic and single-cell transcriptomic analyses revealed that the immune-associated module hub genes were highly expressed in T cells,monocytes and macrophages,as well as the defense-associated module hub genes were highly expressed in endothelial cells in the four-stomach tissues.Moreover,the non-immune cells such as epithelial cells play key roles in immune maturation.Cell commu-nication analysis predicted that in addition to immune cells,non-immune cells recruit immune cells through mac-rophage migration inhibitory factor signaling in the forestomachs.Conclusions Our results demonstrate that the immune and defense responses of four stomachs are quickly devel-oping with age in lamb's early life.We also identified the gene expression patterns and functional cells associated with immune development.Additionally,we identified some key receptors and signaling involved in immune regula-tion.These results help to understand the early life immune development at single-cell resolution,which has implica-tions to develop nutritional manipulation and health management strategies based on specific targets including key receptors and signaling pathways.