3047 – THE ADULT HEMATOPOIETIC STEM CELL NICHE IN ZEBRAFISH DEFINED USING CORRELATIVE LIGHT AND ELECTRON MICROSCOPY

Hematopoietic stem and progenitor cells (HSPCs) that originate from the hemogenic endothelium in the dorsal aorta to finally home and engraft within the adult niche, the fetal bone marrow (BM), become surrounded by niche cells in a complex microenvironment. The ultrastructure of this niche is not well defined as current imaging technology does not allow direct visualization of the fetal BM niche. Zebrafish have a similar hematopoietic ontogeny to mammals, and because the embryos are transparent, intrinsic HSPC interactions with the niche can be directly visualized. To track HSPCs during colonization of the presumptive adult niche, the kidney marrow (KM), we used our previously validated HSPC-specific transgenic reporter lines. In 5 days post fertilization fixed larvae, we detected ∼100 HSPCs/larva. To precisely locate these rare HSPCs within the larger dense KM, we genetically tagged endogenous HSPCs to track them live using lightsheet microscopy, followed by high resolution serial block-face scanning electron microscopy (SBEM). Using this technique, we could visually track single mCherry+ HSPCs, then confirm their exact location in a SBEM dataset with high contrast APEX2 peroxidase label. We found HSPC clusters within vessel lumens, as well as single HSPC in a novel perivascular niche. In this perivascular site, a single HSPC was seen to simultaneously contact one mesenchymal stromal cell, multiple endothelial cells, a glial-like cell and other hematopoietic cells. The glial-like cells were dopamine beta hydroxylase positive (dbh+) cells and treatment with small molecules inhibitor of dopamine led to significantly reduced HSPC numbers within the niche. Our approach can be used to identify the ultrastructure of single rare cells within dense tissues by using multiple imaging modalities and can also lead to the identification of previously uncharacterized cell types.