Figure 3.

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.