Single-cell profiling of goose thymus uncovers the developmental program and regulatory logic of T cell differentiation

The thymus is essential for T cell development and the establishment of central tolerance, yet avian thymic development remains poorly characterized. Using the Zhedong White goose as a model, we analyzed thymic development across four stagesby integrating histological analysis with single-cell RNA sequencing. The goose thymus exhibits a multilobulated architecture, undergoes rapid post-hatch maturation, and shows stage-dependent involution. Single-cell analysis identified the major thymic cell populations, with T cells predominating and ?d T cells present at higher proportions than in mammals. Pseudotime analysis revealed double-positive (DP) cells as a central differentiation hub. Double-negative (DN) thymocytes progressed from DN1 to DN3b through a stepwise program characterized by lineage commitment marked by upregulation of BCL11B, NOTCH1, and TCF7, followed by induction of ß-selection–associated genes, including RAG1/2 and DNTT. Thymic epithelial cells functioned as a major signaling hub, with adhesion- and microenvironment-related pathways coordinating TEC–T cell interactions, while DP and mature T cells progressively emerged as key signal integration nodes. Cross-species comparison indicated an overall conserved developmental framework, with the most pronounced species-specific differences observed at the DP stage. Collectively, this study systematically delineates the developmental trajectories of thymic T cells and their microenvironmental regulation in the goose, providing a valuable resource for studies of avian immune development and comparative immunology. Overall design: To systematically characterize thymic T-cell development in avian species, this study profiled thymic cell populations of female Zhedong White (ZW) geese across four key developmental stages: embryonic day 25 (E25d), post-hatch day 14 (D14), day 70 (D70), and day 200 (D200), corresponding to embryonic thymus formation, early postnatal establishment of immune function, formation of immune homeostasis after sexual maturity, and maintenance of immunity during the laying period, respectively. For single-cell RNA sequencing, thymic tissues were collected from three biologically independent female geese at each developmental stage and pooled to generate one scRNA-seq sample per stage. Thus, each sequencing library represents a pooled biological replicate composed of thymocytes derived from three different individuals at the same developmental stage. To ensure anatomical consistency across samples, the first pair of thymic lobes adjacent to the head was consistently collected from each goose, as the avian thymus is bilaterally distributed along the neck and typically consists of multiple lobes per side. Fresh thymic tissues were preserved in MACS Tissue Storage Solution prior to dissociation, and thymocytes were isolated under sterile conditions for single-cell library preparation and sequencing. In parallel, thymuses from eight additional female geese per stage were used for histological and morphological analyses. Sex was confirmed by PCR amplification of the CHD-1 gene. All animal procedures were approved by the Animal Protection Committee of the Shanghai Academy of Agricultural Sciences (Approval No. SAAS-SL-2023021).