Studies of the avian gastrointestinal tract, where nutrient absorption and key host-pathogen interactions occur, have been strongly enabled by the development of intestinal organoid models. Here we report a single cell transcriptomic atlas of intestinal organoid cells derived from embryos of broiler and layer chickens, capturing mesenchymal, epithelial, endothelial, immune and neuronal cell lineages. Eight inferred mesenchymal subpopulations reflect anatomically distinct intestinal layers, including fibroblasts, telocytes, myofibroblasts, smooth myocytes, pericytes, and interstitial cells of Cajal. Identified heterogeneity within the epithelial lineage included enterocytes, goblet cells, Paneth cells, tuft cells, and diverse enteroendocrine cell subtypes. Additionally, we identified candidate macrophages, monocytes, γδ T cells, NK cells and granulocytes. Layer and broiler organoids showed significant differences in cell-specific transcriptome, most pronounced in epithelial cells, pointing to divergent selection on intestinal physiology. Our analysis finally provides a catalogue of novel cell marker genes to enable future research of chicken intestinal organoids.
© 2025. The Author(s).