T lymphocytes are key cellular components of adaptive immune system in vertebrates. Despite their heterogeneities, it is believed that all different types of T lymphocytes are generated exclusively via the differentiation of hematopoietic stem cells (HSCs). By temporal-spatial resolved fate mapping analysis, here we show that the zebrafish aorta-gonad-mesonephros (AGM) and posterior blood island (PBI), the hematopoietic tissues previously known to generate HSCs and erythro-myeloid progenitors (EMPs), respectively, gives rise to a transient wave of HSC-independent T lymphopoiesis, which occurs early and generates predominantly CD4 T_αβ cells exclusively in the larval stage. In contrast, HSC-derived T lymphopoiesis emerges later and produces various subtypes of T lymphocytes contin...[ Read more ]

T lymphocytes are key cellular components of adaptive immune system in vertebrates. Despite their heterogeneities, it is believed that all different types of T lymphocytes are generated exclusively via the differentiation of hematopoietic stem cells (HSCs). By temporal-spatial resolved fate mapping analysis, here we show that the zebrafish aorta-gonad-mesonephros (AGM) and posterior blood island (PBI), the hematopoietic tissues previously known to generate HSCs and erythro-myeloid progenitors (EMPs), respectively, gives rise to a transient wave of HSC-independent T lymphopoiesis, which occurs early and generates predominantly CD4 T_αβ cells exclusively in the larval stage. In contrast, HSC-derived T lymphopoiesis emerges later and produces various subtypes of T lymphocytes continuously from the larval stage to adulthood. Both two waves of T lymphopoiesis are originated from hemogenic endothelium (HEs) via endothelial-hematopoietic transition (EHT). Further characterization of HEs in zebrafish from 28 hours-post-fertilization shows that the vast majority of ventral endothelium of aorta in both AGM and PBI are hemogenic and undergo EHT before 3 days-post-fertilization, while dorsal wall of aorta contains only ordinary endothelium (OEs) without hemogenic potential, indicating that HEs and OEs are distributed in a spatial-separated manner. Our study unveils the existence, origin and ontogeny of HSC-independent T lymphopoiesis in vivo, as well as systematically characterizes the distribution and dynamics of HEs during embryonic definitive hematopoiesis.