The first wave of T lymphopoiesis in zebrafish arises from aorta endothelium independent of hematopoietic stem cells.

T lymphocytes are key cellular components of the adaptive immune system and play a central role in cell-mediated immunity 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). Using temporal-spatial resolved fate-mapping analysis and time-lapse imaging, here we show that the ventral endothelium in the zebrafish aorta-gonad-mesonephros and posterior blood island, the hematopoietic tissues previously known to generate HSCs and erythromyeloid progenitors, respectively, gives rise to a transient wave of T lymphopoiesis independent of HSCs. This HSC-independent T lymphopoiesis occurs early and generates predominantly CD4 T cells in the larval but not juvenile and adult stages, whereas HSC-dependent T lymphopoiesis emerges late and produces various subtypes of T lymphocytes continuously from the larval stage to adulthood. Our study unveils the existence, origin, and ontogeny of HSC-independent T lymphopoiesis in vivo and reveals the complexity of the endothelial-hematopoietic transition of the aorta.