A Zebrafish Model of Fetal Bone Marrow Provides a Dynamic View of Hematopoietic Stem Cell Niche Colonization

The entire blood system is supported throughout life by a small number of hematopoietic stem and progenitor cells (HSPCs) that are produced exclusively during embryonic development. These stem cells are generated from the hemogenic endothelium of the dorsal aorta, then migrate to the fetal liver where they expand, before making a final migration to the fetal bone marrow. After seeding the bone marrow, the stem cell pool stabilizes and the total number of HSPCs remains relatively constant. Very little is known about this early stage of bone marrow niche colonization. A better understanding of native stem cell pool establishment will likely lead to improved clinical HSPC transplantation that depends on repopulation of the bone marrow niche. Currently, imaging technology does not allow direct visualization of the bone marrow niche during colonization because it occurs in utero in the long bones of the fetus. The zebrafish is an advantageous model because the embryos develop externally and are transparent. To quantify the early stem cell pool, we employed long term fate mapping with clonal analysis using the multicolor Zebrabow system, which imparts a unique fluorescent hue to stem cells and their progeny. Our findings reveal that 21 HSPC clones exist prior to HSPC emergence (24 hours post fertilization) and 30 clones are present during peak production from the aorta (48 hours post fertilization). Seeding of the presumptive adult marrow niche in zebrafish begins 4-5 days post fertilization, versus 16.5 days in the mouse. We previously described a transgenic zebrafish line (Runx:mCherry) that marks long term repopulating HSPCs throughout development and into adulthood. HSPC-specific expression is driven by the well-characterized Runx1 +23 kb mouse enhancer element. We used this line to directly observe the earliest immigration events of HSPCs as they arrive in the marrow. To achieve this, we immobilized the zebrafish by injection of the snake venom protein alpha-bungarotoxin directly into circulation. This allowed long term live imaging of the niche (~16 hours) so we could quantify the dynamics of HSPC colonization and expansion. To rapidly acquire high resolution imaging data for this deep tissue we applied lightsheet microscopy. By simultaneously illuminating the sample in the X plane, while taking images in the Z plane, hundreds of optical sections can be captured in seconds. The high pixel and temporal resolution of lightsheet microscopy in a large volume of tissue provides a highly dynamic view of the entire marrow niche. We could assess the localization of HSPCs in relation to other cell types within the niche. For example, HSPCs were closely associated with endothelial cells in a perivascular niche, similar to what has been described in mammalian bone marrow. Furthermore, we could quantify single Runx+ nuclei over time on one side of the bilateral kidney marrow. During this early stage of niche colonization, we found the number of HSPCs per side was ~50 (so ~100 total) and that remained relatively constant. This was in fact a dynamic equilibrium achieved by ingress and egress of cells, as well as occasional cell divisions. This cell number was independently validated using another transgenic zebrafish line, cd41:GFP, that also marks HSPCs. This quantification, combined with our data from earlier development, suggests that HSPCs undergo around two population doublings between emergence from the aorta and engraftment in the marrow. This unique platform for the quantification of a total stem cell pool will allow further functional and mechanistic studies using both genetics and chemical biology. Our goal is to gain insights into the establishment of the stem cell pool within the niche microenvironment and how this could improve clinical transplantation outcomes.