Figure 4.

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.