Live imaging of emerging hematopoietic stem cells and early thymus colonization

We recently demonstrated in zebrafish the developmental migration of emerging hematopoietic stem cells (HSCs) that is thought to occur in mammalian embryos, from the Aorta-Gonad-Mesonephros (AGM) area to the successive hematopoietic organs. CD41 is the earliest known molecular marker of nascent HSCs in mammalian development. Here we show that in CD41-gfp transgenic zebrafish embryos, the transgene is expressed by emerging HSCs in the AGM, allowing for the first time to image their behavior and trace them in real-time. We find that the zebrafish AGM contains no intra-aortic cell clusters, so far considered a hallmark of HSC emergence. CD41-GFP(low) HSCs emerge in the sub-aortic mesenchyme, and enter the circulation not through the dorsal aorta but through the axial vein, whose peculiar structure facilitates their intravasation. The rise in CD41-gfp expression among c-myb+ HSC precursors is asynchronous, and marks their competence to leave the AGM and immediately seed the Caudal Hematopoietic Tissue (which has a hematopoietic function analogous to the mammalian fetal liver). Imaging the later migration of CD41-GFP+ precursors to the nascent thymus reveals that although some reach the thymus by extravasating from the nearest vein, most travel for hours through the mesenchyme, from surprisingly diverse and remote sites of extravasation.