Summary of the main steps of the RNAscope procedure.

AMP: signal AMPlifier (1, 2, or 3); HRP: HorseRadish peroxidase; HRP-C: HRP channel (1, 2, or 3); TSA: tyramide signal amplification; ZZ: pair of oligonucleotide probes [with target-specific binding sites covering regions of approximately 50 bases and mRNA target regions covering 300–1,000 nucleotides (with a pool of 6–20 ZZ pairs)]. OR + grey arrow indicates, on Day 2, the repeat of steps involved in additional fluorophore coupling using HRP activity in case of probe multiplexing using other channels (two times if two probes, three times if three probes) or interruption of the procedure until mounting of embryos/larvae on Day 3.

Efficiency of proteinase K digestion on the zebrafish embryo and larva.

48 hpf embryos (a) and 5 dpf larvae (b) were enzymatically digested or not (+ proteinase K, - proteinase K, respectively) after formaldehyde and methanol fixation. Note the effect of proteinase K treatment that is visualized by an increase in transparency of both the embryo and larva. In the embryo, the brain also appears more “milky,” which indicates the extreme limit of acceptable digestion.

Whole-mount in situ analysis of cmyb mRNA expression in a 48 hpf embryo

. Top panel: schematic representation of a 48 hpf embryo (reproduced with modifications from [16]); red line = aorta (from which hematopoietic stem cell precursors emerge); blue line = vein (showing particularly the vein plexus, constituting the CHT). The embryo used expresses eGFP under the control of the vascular kdrl promotor [Tg(kdrl:eGFP) fish line]. Bottom panels: spinning disk confocal images obtained either in the trunk region (left: AGM) or in the tail region (right: CHT). Images shown were obtained either from maximal z-projections of z-stacks or from single z-sections with 3× magnification of regions in white boxes. Green channel: vascular cells expressing soluble eGFP [aortic and vein endothelial cells, including cells of the hemogenic endothelium (HE; white arrows) in the trunk and tail regions] as well as newly born HSPCs (white asterisks). Magenta: RNAscope spots indicating expression of cmyb mRNAs in the HE and in HSPCs. Note the presence of RNAscope spots in the trunk (top left image) and in the gut region (magenta arrows) that indicate the potential expression of cmyb in more differentiated patrolling immune cells. AGM: aorta gonad mesonephros; CHT: caudal hematopoietic tissue; HSPCs: hematopoietic stem and progenitor cells. Scale bars = 20 μm.

Whole-mount in situ analysis of cmyb mRNA expression in a 5 dpf larva

. Top panel: schematic representation of a 5 dpf larva (reproduced with modifications from [16]); red line = aorta; blue line = vein. The larva used expresses eGFP under the control of the vascular kdrl promotor [Tg(kdrl:eGFP) fish line]. Bottom panels: spinning disk confocal images obtained either in the trunk region (left: AGM) or in the tail region (right: CHT). Images shown were obtained either from maximal z-projections of z-stacks or from single z-sections with 1.5× magnification of regions in white boxes. Green channel: vascular cells expressing soluble eGFP (aortic and veinous endothelial cells in the trunk and tail regions). Magenta: RNAscope spots indicating expression of cmyb mRNAs in HSPCs. Note that RNAscope signals cannot be superposed to individual cells because HSPCs are no longer expressing eGFP driven by the vascular kdrl promoter, as is the case in 48 hpf embryos (owing to distant timing from their emergence, half-life of eGFP, and division cycles diluting the fluorescent protein). Note the structural evolution of the vein niche, notably in the CHT, in comparison with the 48 hpf embryo shown in Figure 3, and the relatively regular positioning of hematopoietic clusters (delimited by dashed lines in the bottom right image). AGM: aorta gonad mesonephros; CHT: caudal hematopoietic tissue; HSPCs: hematopoietic stem and progenitor cells. Scale bars = 20 μm.

Whole-mount in situ analysis of RNAscope signals deep in the pronephros region of 5 dpf larvae using the cmyb probe.

Spinning disk confocal images obtained with a 5 dpf larva [Tg(kdrl:eGFP) fish line] expressing eGFP in endothelial cells under the control of the vascular kdrl promotor. Images show the pronephros region with a maximal z-projection (left) of a z-stack encompassing a total of 240 sections, each interspaced by 0.5 μm, and z-sections (right: images of sections 52/240, 86/240, 186/240, and 222/240 from top to bottom, respectively). The cmyb RNAscope signals (magenta) correspond to hematopoietic stem and progenitor cells (HSPCs) accumulated in the peri-glomerular region (white arrow), surrounded by vessels. Note the more distant localization of cmyb signals (magenta arrows, hypothetically in HSPCs) along vessels (green arrows). This unveils the potential complexity of the pronephros niche. ISV = intersegmental vessel. This z-stack is also visualized in Video 1 and reconstituted in 3D using Imaris in Video 2. Note also the nonspecific capture of the dye in the notochord, which facilitated the localization of the glomerulus. Scale bars = 20 μm.

Whole-mount in situ analysis of cmyb mRNA colocalization with HSPCs at 5 dpf.

Spinning disk confocal images obtained with a 5 dpf larva [Tg(runx1+23:eGFP) fish line] expressing eGFP in HSPCs under the control of the runx1+23 enhancer. Panels are constituted from images obtained either in the trunk region (left: AGM) or in the tail (right: CHT), either from maximum z-projections of z-stacks or from single z-sections with 2× magnification of regions in white boxes, highlighting hematopoietic clusters (dashed lines). RNAscope signals (magenta) colocalize in the majority with HSPCs (see also Figure 7 and Video 3 for 3D reconstitution). AGM: aorta gonad mesonephros; CHT: caudal hematopoietic tissue; HSPCs: hematopoietic stem and progenitor cells. Scale bars = 20 μm.

Cell and RNAscope segmentation workflow with Imaris.

All data shown are extracted from a single caudal hematopoietic tissue (CHT) z-stack showing the expression of cmyb (RNAscope, spots in magenta) in hematopoietic cells expressing eGFP under the control of the runx1+23 enhancer (the same 5 dpf larva and images as in Figure 6). (A) File conversion with Imaris File Converter. (B) Imaris 3D visualization in surpass mode. (C) Step-by-step workflow of cell segmentation, as detailed in Data Analysis, Section B. 3D cells and RNAscope signal segmentation with Imaris – Cell Biologist package.

Mounting of embryos and larvae after RNAscope reagents treatment.

Top images show Ibidi dishes, with the recommended dissection area on the top and the agarose mounting area at the bottom (white rectangles). 48 hpf embryos (a, b) and 5 dpf larvae (a’, b’) were either (a, a’) or not (b, b’) treated with proteinase K and all of them with RNAscope reagents. Images show that the whole procedure leads to virtually total transparency of the specimen, which reaches a maximum for embryos [becoming barely visible as in (a)]. This transparency requires controlling all steps of reagent/buffer removal under the binoculars. Yolks were dissected, as well as the eyes facing the glass bottom of the dish to ensure flatness.

Spatial analysis and plotting of gene expression data.

All data plotted are extracted from a single caudal hematopoietic tissue (CHT) z-stack showing the expression of cmyb (RNAscope spots) in hematopoietic cells expressing eGFP under the control of the runx1+23 enhancer. (A) Imaris Vantage 2D plotting, cell number of RNAscope spots relative to the cell area (µm). (B) Imaris vantage cumulative distribution function (CDF) plotting, cumulative number of spots percentage relative to the shortest distance to cell surface (µm). Solid pink line: our data; dashed pink line and light pink interval: confidence interval’s random distribution; red vertical line: calculated attraction distance. (C) Data extraction from Imaris and plotting in R studio using the ggstatsplot package. Top plot shows the correlation between cell size (µm) and number of spots per cell. Bottom plot shows the number of spots per cell depending on cell size [large cell > 11,490 voxels (corresponding to the median cell size), small cells < 11,490 voxels]. The code to generate the plots is given in Data analysis, section C.

RNAscope in situ cmyb expression analysis in the pronephros region at 5 dpf.

Spinning disk confocal images (z-stack) obtained from a 5 dpf Tg(kdrl:eGFP) larva. The eGFP is expressed in endothelial cells under the control of the vascular kdrl promotor. Z-stack shows the pronephros region; consecutive confocal z-sections are 0.5 μm interspaced. The cmyb RNAscope signals (magenta) are localized in hematopoietic stem and progenitor cells (HSPCs) accumulated in the peri-glomerular region, surrounded by vessels. Unique z-sections (52/240, 86/240, 186/240, and 222/240) and maximum z-projection are also shown in Figure 5. This z-stack is also reconstituted in 3D in Video 2.

Imaris 3D reconstitution of RNAscope in situ cmyb expression analysis in the pronephros region at 5 dpf.

3D rendering (reconstituted with the Imaris software) obtained from a spinning disk confocal image (z-stack) obtained from a 5 dpf Tg(kdrl:eGFP) larva. The eGFP (green) under the control of the kdrl vascular promoter is expressed in endothelial cells. Z-stack shows the pronephros region. The cmyb RNAscope signals (magenta) are localized in hematopoietic stem and progenitor cells (HSPCs) accumulated in the peri-glomerular region, surrounded by vessels. Unique z-sections and maximum z-projection are also shown in Figure 5, and the complete z-stack is shown in Video 1.

Imaris 3D rendering and colocalization of RNAscope spots and hematopoietic stem and progenitor cells (HSPCs) in the CHT region.

3D reconstitution generated from a spinning disk confocal z-stack obtained from a 5 dpf Tg(runx1+23:eGFP) larva. eGFP (green) under the control of the runx1+23 enhancer is expressed in HSPCs. RNAscope signals (magenta) colocalize in the majority with HSPCs (see also Figure 6 for maximum z-projections and single z-sections and Figure 7 for 3D reconstitution and cell segmentation steps).