Characterizing the Adult Hematopoietic Stem Cell (HSC) Niche in a Zebrafish Model for Fetal Bone Marrow

Hematopoietic stem and progenitor cells (HSPCs) originate from the dorsal aorta and migrate to the intermediate niche, the fetal liver, where they undergo rapid expansion before migrating to the prospective adult niche, the bone marrow. This early stage of bone marrow colonization is poorly understood. Current imaging technology does not allow direct visualization of the fetal bone marrow niche. Zebrafish embryos can be used to directly visualize HSPC‐niche cell interactions unperturbed because they develop externally and are transparent. Importantly, their HSC ontogeny is highly similar to mammals. The zebrafish kidney is equivalent to the adult bone marrow niche and gets colonized from 4 to 5 days post fertilization (dpf). To track HSPCs during this dynamic process, we used our previously characterized HSPC‐specific transgenic reporter lines (Runx:GFP and Runx:mCherry). We demonstrated by transplantation that these cells mark a highly pure population of long‐term engrafting hematopoietic stem cells. Using a tissue clearing technique that allows visualization of deeper tissues, we detected ~100 HSPCs in the kidney marrow in 5 dpf embryos. These cell numbers could be manipulated through drug treatments and gene knockdowns. For live‐imaging of the kidney marrow, we applied lightsheet microscopy that allows rapid image acquisition with high spatio‐temporal resolution, providing a highly dynamic view of the entire developing niche. By imaging Runx+ HSPC together with flk+ endothelial cells in a double transgenic embryo, we could track interactions between HSPC and the perivascular niche. Previously, we showed that HSPC arrival in the caudal hematopoietic tissue, the zebrafish equivalent of the fetal liver, triggers remodeling of endothelial cells into a surrounding pocket. In this study, we found that a similar cellular structure forms within the kidney marrow niche. To resolve the ultrastructure of the HSPC and its surrounding cells in context of the entire niche, we performed Correlative Light and Electron Microscopy. For this, the embryos imaged on lightsheet were fixed and stained for EM. MicroCT was used to orient and trim the embedded sample to isolate the kidney niche and serial blockface scanning EM to capture high resolution serial sections. By matching anatomical markers such as blood vessels observed in light and EM 3D data sets, a single round HSPC with a large nucleus, little cytoplasm, and a ruffled membrane, was identified lodged just under the vessel wall. This single HSPC was in a distinct anatomical location compared to larger clusters of HSPC in an abluminal space. This is suggestive of distinct anatomical niche sites within the marrow that may support different functions, such as proliferation versus quiescence. Our goal is to gain novel insights into intercellular interactions between HSPC and the surrounding supportive cells in the perivascular niche.Support or Funding InformationASH, NIDDKThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.