Genetic Analyses in Zebrafish Reveal That Angiopoietin-Like Proteins 1 and 2 Are Required for HSC Development during Embryogenesis

Recent studies have shown that members of the angiopoietin-like protein (angptl) family can help stimulate ex vivo expansion of mouse and human hematopoietic stem cells (HSCs). The requirement of angptl for HSC development has yet to be determined. Here, the effects of morpholino (MO) knockdown of angptl1, angptl2 or the combination of both, were assessed during zebrafish development. Single MO knockdown of either angptl1 or angptl2 resulted in a decrease in c-myb positive HSCs in the aorta-gonad-mesonephros (AGM) region, the site of definitive hematopoiesis at 36 hours post-fertilization (hpf). Embryos injected with combined angptl1 and angptl2 MOs (double morphants) had an even more severe phenotype, in which both c-myb and runx1 positive HSCs were almost completely absent. In an effort to discern the onset of the phenotype, early HSC markers were evaluated. The double morphants had decreased expression of scl and lmo2 at 8–10 somite stages (ss) in the bilateral stripes of the lateral mesoderm region from which hematopoietic and vascular progenitors are thought to arise. To determine if angptl are sufficient to expand HSCs, angptl1 and angptl2 mRNA were injected into one-cell stage embryos and HSC markers were evaluated. Angptl1 and angptl2 mRNA significantly increased c-myb and runx1 positive HSCs in the AGM. Interestingly, addition of thrombopoietin, a potent stem cell growth factor, with angptl1 and angptl2 further increased HSCs in the AGM, implying a synergistic effect of these growth factors during HSC development. Angptl regulation of HSC development may occur by stimulating the production of the hemogenic endothelium. MO knockdown of either angptl1 or angptl2 alone did not alter vascular development significantly, while MO knockdown of both angptl1 and angptl2 resulted in a disruption of intersegmental blood vessel sprouting at 28 hpf. The arterial and venous specification in these double morphants was also disrupted at 28 hpf, with decreased expression of the arterial marker ephrinB2 but increased ectopic expression of the venous marker, flt4. These results strongly suggest an early downstream effect of angptl signaling on hemogenic endothelium. Furthermore, since Vegf signaling is indispensable for proper endothelial specification and definitive HSC formation, we found that in the double morphants, somite expression of vegf₁₆₅ mRNA was decreased early in development. Although angptl1 mRNA expression was undetectable at this stage, angptl2 mRNA expression was localized in the yolk sac extension and posterior spinal cord, appearing in close proximity to the vegf positive somites. This suggests that in these locations, angptl may stimulate Vegf production in the embryo to participate in the induction of hemogenic endothelium and subsequently lead to HSC formation. Taken together, our data demonstrates that the angptl are required for HSC development by coordinating the production of a functional hemogenic endothelium that can lead to the formation of HSCs.