Figure 5.

Figure 5. 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.