ABSTRACT: Understanding and manipulating the avian gastrointestinal tract, where nutrient absorption and vital host-pathogen interactions occur, is critical for improving poultry health and productivity. To advance research in this area, chicken three-dimensional intestinal enteroid models have been developed and characterised. In this study, we report a comprehensive transcriptome atlas for 43,587 intestinal enteroid cells derived from broiler and layer chickens. Our analyses reveal clusters representing mesenchymal, epithelial, endothelial, immune and neuronal cell lineages. We classified the mesenchymal lineage into nine populations, including fibroblasts, telocytes, myofibroblasts, smooth myocytes, pericytes, and interstitial cells of Cajal, reflecting anatomically distinct intestinal layers. We identified significant heterogeneity within the epithelial lineage, including enterocytes, goblet cells, Paneth cells, tuft cells, and various enteroendocrine cell subtypes. Additionally, we provide evidence for populations of macrophages, monocytes, γδT cell, NK cells, heterophils and dendritic cells. Differential gene expression tests between layer and broiler enteroids revealed extensive cell type-specific variation, pointing at differences in immune function and appetite regulation, with the most pronounced changes in epithelial cell types. Overall, this study offers a cell-resolved perspective of chicken three-dimensional intestinal enteroids, uncovering extensive heterogeneity masked by bulk transcriptomics. Additionally, we provide a catalogue of cell-specific marker genes to explore the function and organization of the chicken gastrointestinal tract.
