Correlative Lightsheet and Electron Microscopy Applied to a Zebrafish Model of Fetal Bone Marrow: Resolving the Ultrastructure of a Single Hematopoietic Stem Cell in the Niche

Hematopoietic stem and progenitor cells (HSPC) emerge from the hemogenic endothelium of the dorsal aorta. Next, HSPC migrate to the fetal liver where they expand exponentially before migrating again to the fetal 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 provide a model to observe these events because they develop externally, are transparent, and ontogeny of the hematopoietic system is highly conserved. The adult marrow niche in zebrafish is in the kidney and is colonized 4 to 5 days post fertilization. To track HSPC during this dynamic process we used our previously characterized HSPC-specific transgenic reporter lines (Runx:GFP and Runx:mCherry). We demonstrated by transplantation these cells mark a highly pure population of long-term engrafting hematopoietic stem cells. To image the kidney marrow live we applied lightsheet microscopy. This technology uses three objective lenses: two that illuminate the sample in the XY plane, and one that takes images in the Z plane. This allows hundreds of optical sections to be captured in seconds. The temporal and spatial resolution of this technique produces a highly dynamic view of the entire developing marrow 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 a similar cellular structure forms in the kidney marrow niche. We wanted to resolve the ultrastructure of the HSPC and surrounding cells in the context of the larger developing kidney organ. To do this we applied Correlative Light and Electron Microscopy (CLEM) to match fluorescent cells tracked by lightsheet in the same embryo fixed and stained for EM. Micro-CT was used to orient and trim the embedded sample to isolate the kidney niche. Serial block-face scanning EM was used to capture high resolution serial sections (volume dimensions: x, y, z (um) = 236, 114, 105; pixel size: x, y, z (nm) = 9.5, 9.5, 70). Blood vessels observed in light and EM 3D data sets provided the anatomical markers needed to match the position of cells found using both techniques. As we previously found in the caudal hematopoietic tissue, a single round HSPC with a large nucleus, little cytoplasm, and a ruffled membrane, was 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 observation is suggestive of a marrow with distinct anatomical niche sites that may support different functions, such as proliferation versus quiescence. This unique system for analysis of the ultrastructure of an endogenous HSPC in an unperturbed niche will allow further functional and mechanistic studies at the subcellular level. Our goal is to gain novel insights into intercellular interactions between HSPC and the surrounding supportive cells in the perivascular niche.