Defining the ultrastructure of the hematopoietic stem cell niche by correlative light and electron microscopy
- Center for Stem Cell and Regenerative Medicine, Department of Pharmacology, College of Medicine, University of Illinois at Chicago, United States
- Center for Research in Biological Systems, National Center for Microscopy and Imaging Research, University of California at San Diego, United States
- Department of Neurosciences, University of California at San Diego School of Medicine, United States
- Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin-Madison, United States
Figures
Figure 1 with 7 supplements
Single hematopoietic stem and progenitor cells (HSPCs) lodge in a perivascular region of the larval kidney niche.
(A) Snapshot of single optical sections (XY, XZ, YZ planes) from light sheet live image of a Runx:GFP;flk:mCherry double transgenic zebrafish larva. A single Runx:GFP+ HSPC (white arrowhead) is …
Figure 1—figure supplement 1
Quantification of Runx:mCherry+ HSPCs in the anterior kidney of the zebrafish larva.
(A) Single Z plane of a confocal stack of fixed 5 dpf Runx:mCherry+ (magenta) transgenic zebrafish larva imaged after optical tissue clearing. The location of glomerulus (G, dotted blue circle) and …
Figure 1—figure supplement 2
First frame from light sheet live imaging of Runx:mCherry+ 105 hr post fertilization (hpf) larva.
Corresponds to Figure 1—video 1. Depth-coded projection in Z plane from left (0 μm red < orange < yellow <green) to right (blue < indigo < violet 150 μm). Runx:mCherry+ hematopoietic stem and …
Figure 1—figure supplement 3
Light sheet live imaging shows hematopoietic stem and progenitor cell (HSPC) clusters are mediolateral to the proximal pronephric tubules (PT).
Larva is 5 days post fertilization (dpf). (Ai–Aiii) Lateral and (Aiv–Avi) dorso-lateral views of Runx:mCherry and cdh17:GFP double transgenics labeling the HSPCs and PT, respectively. HSPC cluster …
Figure 1—figure supplement 4
Light sheet live imaging of the anterior kidney reveals hematopoietic stem and progenitor cells (HSPCs) in distinct perivascular regions.
Larva is 5 days post fertilization (dpf). (A) (i) 3D projection of the anterior kidney in Runx:mCherry+ (HSPCs) and flk:ZsGreen+ (ECs) double transgenic larva imaged using light sheet microscope. …
Figure 1—figure supplement 5
Light sheet live imaging of overlap of hematopoietic stem and progenitor cell (HSPC) transgenic reporter lines.
(A) Runx:mCherry and cd41:GFP double positive larva (4 days post fertilization [dpf]). (i) 3D projection shows overlap between the Runx:mCherry and cd41:GFP positive populations (white dashed …
Figure 1—video 1
Depth-coded projection of a time-lapse video shows migrating mCherry+ hematopoietic stem and progenitor cells (HSPCs) occupying the kidney marrow (KM) niche.
Corresponds to Figure 1—figure supplement 2. Runx:mCherry+ zebrafish larva embedded in low melting agarose was aligned to the laser path from the left side to illuminate the KM, which results in a …
Figure 1—video 2
Single circulating hematopoietic stem and progenitor cells (HSPCs) interact with the posterior perivascular niche region.
Corresponds to Figure 1. Time-lapse video shows Runx:GFP+ HSPCs (green) in circulation through flk:mCherry+ vessels (magenta). Occasionally, HSPCs slow and attach to vessels in the perivascular …
Figure 2
Hematopoietic stem and progenitor cells (HSPCs) lodged in the larval kidney niche make direct contacts with endothelial cells (ECs) and mesenchymal stromal cells (MSCs).
(A) Single optical section from confocal image of larval kidney (fixed) shows Runx:mCherry+ HSPCs (magenta) lodged in the perivascular niche. Oregon Green dye labels the vessel lumen. Blue dotted …
Figure 3 with 4 supplements
Correlative light and electron microscopy (CLEM) Workflow #1 to genetically encode a label in endogenous hematopoietic stem and progenitor cells (HSPCs) for live tracking by light microscopy and high-contrast resolution in serial block-face scanning electron microscopy (SBEM) sections.
(A) Fusion construct encoding p2A-linked proteins mito-APEX2, APEX2-H2B, and mCherry that localize to the mitochondria, nucleus, and cytoplasm, respectively. The draculin promoter was used to …
Figure 3—figure supplement 1
Single cd41:GFP+ hematopoietic stem and progenitor cell (HSPC) lodged in the perivascular niche.
(A) Snapshot of single optical sections (XY, XZ, YZ planes) from light sheet live image of a 5 days post fertilization (dpf) cd41:GFP;flk:mCherry double transgenic zebrafish larva. A single cd41:GFP+…
Figure 3—figure supplement 2
Method for mounting, aligning, and trimming the embedded sample for serial block-face scanning electron microscopy (SBEM).
(A) The zebrafish tissue block on the aluminum pin is completely opaque to light following staining for SBEM. (B) Micro-computed tomography (microCT) reveals details within the embedded block and …
Figure 3—video 1
Transiently labeled hematopoietic stem and progenitor cells (HSPCs) lodge within the perivascular niche.
Corresponds to Figure 3. Time-lapse movie shows a single lodged mCherry+ HSPC (magenta; yellow arrowhead) in a transient drl:APEX2-mCherry+-injected larva. The cell is lodged ventral to the DA and …
Figure 3—video 2
Micro-computed tomography (microCT) stack acquired through a fixed zebrafish larva.
Corresponds to Figure 3. MicroCT is an intermediate step between light sheet imaging and serial block-face scanning electron microscopy (SBEM) that is used to orient the sample and define our region …
Figure 4 with 4 supplements
3D alignment of light sheet and serial block-face scanning electron microscopy (SBEM) datasets localizes a single rare hematopoietic stem and progenitor cell (HSPC) across multiple imaging modalities.
(A) Single Z plane from light sheet imaging of drl:APEX2-mCherry+ transgenic larva showing the lodged mCherry+ HSPC (white arrowhead). (B) Global alignment of 3D rendered models generated from light …
Figure 4—figure supplement 1
Segmentation and 3D surface rendering identifies anatomical features within the serial block-face scanning electron microscopy (SBEM) datasets.
(A) (i) Schematic dorsal view of a zebrafish larva. (ii) Oblique slice through 3D SBEM data shows the right (green) and left (purple) pronephric tubules, glomerulus (blue), and the right (red) and …
Figure 4—figure supplement 2
Tracing of serial block-face scanning electron microscopy (SBEM) shows location of anterior hematopoietic stem and progenitor cell (HSPC) clusters relative to the glomerulus, pronephric tubules, and cardinal veins.
(A) (i) Oblique slice along the transverse view of 3D SBEM of zebrafish larva shows the location of anterior HSPC clusters relative to the pronephric tubules, cardinal veins, and the glomerulus. …
Figure 4—figure supplement 3
Alignment of 3D rendered models from serial block-face scanning electron microscopy (SBEM) and light sheet live imaging shows anatomical features in all three planes.
(A) Parasagittal view shows somites, pneumatic duct, intestine, sinus venosus, and inter-segmental vessels (ISVs). (B) Sagittal view shows glomerulus, dorsal aorta, and intestines. (C) Frontal view …
Figure 4—video 1
All sections (>3000) of a APEX2+ serial block-face scanning electron microscopy (SBEM) dataset.
Figure 5 with 4 supplements
Hematopoietic stem and progenitor cells (HSPCs) lodge in a multicellular niche in the perivascular kidney marrow (KM).
The ultrastructure of a single APEX2+ HSPC (white arrow) and its surrounding niche cells are modeled using 3D SBEM (00:15 from Figure 4—video 1). (A) The APEX2+ HSPC is lodged in the perivascular KM …
Figure 5—figure supplement 1
Hematopoietic clusters are visible in the serial block-face scanning electron microscopy (SBEM) dataset.
A different section of the SBEM dataset shown in Figure 5 shows the ultrastructure of the hematopoietic clusters relative to the surrounding tissues (00:17 from Figure 4—video 1). Abbreviations: D, …
Figure 5—figure supplement 2
3D alignment of a second serial block-face scanning electron microscopy (SBEM) dataset showing localization of a single hematopoietic stem and progenitor cell (HSPC) across multiple imaging modalities.
Five days post fertilization (dpf) larva. (A) Location of the mCherry+;APEX2+ HSPC (white arrow) in the posterior perivascular niche region, relative to anatomical features such as somites, …
Figure 5—figure supplement 3
Tracing of unlabeled putative hematopoietic stem and progenitor cells (HSPCs) in a second serial block-face scanning electron microscopy (SBEM) dataset shows their relationship with niche cells.
Five days post fertilization (dpf) larva. The ultrastructure of two putative APEX2- HSPCs (based on morphology), and their surrounding niche cells, are modeled using 3D SBEM. The morphological …
Figure 5—video 1
Hematopoietic stem and progenitor cells (HSPCs) lodge in a multicellular niche structure in the perivascular kidney marrow (KM).
Corresponds to Figure 5. Orthogonal views through the serial block-face scanning electron microscopy (SBEM) data in all three planes shows the dense tissue in the larval KM region. APEX2+ staining …
Figure 6 with 2 supplements
Correlative light and electron microscopy (CLEM) Workflow #2 to align all cd41:GFP+ hematopoietic stem and progenitor cells (HSPCs) in the larval kidney marrow (KM) niche.
Five days post fertilization (dpf) cd41:GFP+ HSPCs (green) and DRAQ5 nuclear dye (blue). (A-D) The same region of the KM niche is marked by a white dotted rectangle. (A) Confocal and brightfield …
Figure 6—figure supplement 1
Positional overlap of cd41:GFP+ hematopoietic stem and progenitor cells (HSPCs) in confocal data and segmentation of putative HSPCs in serial block-face scanning electron microscopy (SBEM) data.
(A) Z projection of cd41:GFP+ HSPCs (green) and DRAQ5+ nuclei (blue) surrounded by larger grid box (xy orientation). Smaller grid box shows aligned position of SBEM dataset (see Figure 6D). (A’) …
Figure 6—figure supplement 2
Segmentation of cd41:GFP+ and cd41:GFP- putative hematopoietic stem and progenitor cells (HSPCs) in serial block-face scanning electron microscopy (SBEM) data.
(A) Defined GFP+ objects in 3D confocal volume (HSPC #1–10; see Figure 6—figure supplement 2). Left panel is a detail of one section from the SBEM dataset. Right panel shows overlay of aligned …
Figure 7 with 3 supplements
Dopamine beta-hydroxylase positive ganglion-like cells are present within the larval kidney marrow niche.
(A) 3D rendering generated using light sheet movies of Runx:mCherry;dbh:GFP double transgenic larva shows mCherry+ clusters (dotted ovals) in close proximity to GFP+ cells. DA, dorsal aorta. (B) …
Figure 7—figure supplement 1
Correlative light and electron microscopy (CLEM) confirms that dbh:GFP+ cells are present within the kidney marrow (KM) and neurotoxin targeting dopaminergic cells reduces hematopoietic stem and progenitor cell (HSPC) colonization of the KM niche.
(A) Quantification of distances measured between dbh:GFP+ cells and Runx:mCherry+ HSPCs shows that almost 30% of HSPCs are in contact with dbh:GFP+ cells and the remaining are within 13 µm. Numbers …
Figure 7—video 1
Alignment of confocal and micro-computed tomography (microCT) stacks for the dbh:gfp+ larvae.
Corresponds to Figure 7. Aligned sections through the correlated stack in XY plane. MicroCT (grayscale), confocal dbh:gfp+ (green), confocal DRAQ5 nuclei (blue). Magnification ×20. Scale bar: 50 µm. …
Figure 7—video 2
Correlative light and electron microscopy (CLEM) of confocal and serial block-face scanning electron microscopy (SBEM) datasets confirms dbh:gfp+ cells form part of the larval kidney marrow (KM) niche.
Corresponds to Figure 7. Aligned sections through the correlated stack of confocal and SBEM data in the XY plane shows that dbh:GFP+ cells are located within the KM niche. Rendered at 10 frames per …
Tables
Table 1
Summary of all putative hematopoietic stem and progenitor cells (HSPCs) in serial block-face scanning electron microscopy (SBEM) datasets, their locations, and niche cell contacts.
‘Cell Image Library (CIL)’ refers to the public database accession numbers. ‘SBEM dataset’ refers to one of the four datasets used in the study: APEX2 #1, APEX2 #2, cd41:GFP, and dbh:GFP. ‘Figure’ …
| Cell Image Library (CIL) | SBEMdataset | Figure | PutativeHSPC | Label | Color code | Location | EC contact | MSC contact | RBC contact | HSPC contact | GL contact |
|---|---|---|---|---|---|---|---|---|---|---|---|
| CIL:54847 | APEX2 #1 | 5 | 1 | APEX2+ | Green | Perivascular | 5 | 1 | 0 | 2 | 1 |
| CIL:54846 | APEX2 #2 | 5-S2 | 1 | APEX2+ | None | Lumen | 2 | 0 | 0 | 0 | 0 |
| CIL:54846 | APEX2 #2 | 5-S3 | 1 | APEX2- | Green | Perivascular | 5 | 1 | 0 | 0 | 1 |
| CIL:54846 | APEX2 #2 | 5-S3 | 2 | APEX2- | Green | Perivascular | 5 | 1 | 0 | 0 | 0 |
| CIL:54849 | cd41:GFP | 6 | 1 | GFP+ | Red | Cluster | 5 | 0 | 0 | 0 | 0 |
| CIL:54849 | cd41:GFP | 6 | 2 | GFP+ | Orange | Lumen | 3 | 0 | 3 | 0 | 0 |
| CIL:54849 | cd41:GFP | 6 | 3 | GFP+ | Yellow | Cluster | 5 | 1 | 2 | 2 | 0 |
| CIL:54849 | cd41:GFP | 6 | 4 | GFP+ | Lime | Cluster | 3 | 0 | 1 | 0 | 0 |
| CIL:54849 | cd41:GFP | 6 | 5 | GFP+ | Bright green | Cluster | 5 | 1 | 0 | 0 | 0 |
| CIL:54849 | cd41:GFP | 6 | 6 | GFP+ | Teal | Cluster | 3 | 0 | 3 | 0 | 0 |
| CIL:54849 | cd41:GFP | 6 | 7 | GFP+ | Light blue | Cluster | 5 | 0 | 1 | 0 | 0 |
| CIL:54849 | cd41:GFP | 6 | 8 | GFP+ | Dark blue | Lumen | 3 | 0 | 1 | 0 | 0 |
| CIL:54849 | cd41:GFP | 6 | 9 | GFP+ | Magenta | Lumen | 3 | 0 | 2 | 0 | 0 |
| CIL:54849 | cd41:GFP | 6 | 10 | GFP+ | Dark red | Cluster | 5 | 1 | 1 | 1 | 0 |
| CIL:54849 | cd41:GFP | 6 | 11 | GFP low | White | Cluster | 2 | 1 | 1 | 3 | 0 |
| CIL:54849 | cd41:GFP | 6 | 12 | GFP low | White | Cluster | 3 | 1 | 1 | 0 | 0 |
| CIL:54849 | cd41:GFP | 6 | 13 | GFP low | White | Cluster | 6 | 0 | 1 | 0 | 0 |
| CIL:54849 | cd41:GFP | 6 | 14 | GFP low | White | Cluster | 4 | 1 | 1 | 0 | 0 |
| CIL:54849 | cd41:GFP | 6 | 15 | GFP- | White | Cluster | 5 | 0 | 1 | 1 | 0 |
| CIL:54849 | cd41:GFP | 6 | 16 | GFP- | White | Cluster | 5 | 0 | 2 | 0 | 0 |
| CIL:54849 | cd41:GFP | 6 | 17 | GFP low | White | Cluster | 5 | 1 | 2 | 0 | 0 |
| CIL:54848 | dbh:GFP | 7 | 1 | None | Magenta | Perivascular | 5 | 1 | 0 | 1 | 1 |
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EC, endothelial cell; MSC, mesenchymal stromal cell; RBC, red blood cell; HSPC, hematopoietic stem and progenitor cell; GL, ganglion-like cell.
