Genetic Interaction Between Angiopoietin-Like Proteins 1 and 2 and Notch Signaling During HSC Development

Abstract 573

Members of the angiopoietin-like protein (angptl) family have recently been shown to stimulate ex vivo expansion of mouse and human hematopoietic stem cells (HSCs). The requirement of angptls for HSCs during development and their mechanism of action have yet to be determined. To first recapitulate the effects of exogenous angptl on HSC expansion, we made a stable transgenic zebrafish line, Tg(hsp70:angptl2), that overexpresses angptl2 upon heatshock induction. Heatshocked Tg embryos showed a significant increase in cmyb- and runx1-positive HSCs in the aorta-gonad-mesonephros (AGM) region, the site of definitive hematopoiesis at 36 hours post-fertilization (hpf), suggesting that angptl2 can sufficiently expand definitive HSCs during development. We then assessed the effects of angptl1 and/or angptl2 morpholino (MO) knockdown. Single MO knockdown resulted in a decrease in cmyb- and runx1-positive HSCs in the AGM whereas embryos injected with combined angptl1 and 2 MOs (double morphants) exhibited more severe phenotype in which HSCs were completely absent. Angptl regulation of HSC development may occur by stimulating the production or specification of a patent hemogenic endothelium, as the double morphants displayed a disruption of vascular specification at 28 hpf, with decreased expression of the arterial marker ephrinB2 but increased ectopic expression of the venous marker, flt4 in the dorsal aorta. These double morphants also exhibited disrupted intersegmental blood vessel sprouting. Because proper patterning of the developing blood vessels is a prerequisite for subsequent HSC formation, these results strongly suggest an early downstream effect of angptl signaling on hemogenic endothelium specification. To dissect the mechanism of angptl signaling, we asked whether there is a genetic interaction between notch and angptl signaling since the observed phenotype in the angptl double morphants resembled that in the notch mutant mindbomb, mib. We first monitored notch signaling using a transgenic notch reporter zebrafish line that expresses EGFP when the active component of notch signaling, notch intracellular domain (NICD), is expressed. MO knockdown of angptls 1 and 2 resulted in an absence of notch signaling, particularly in the vasculature. Interestingly, overexpressing angptl2 by crossing Tg(hsp70:angptl2) into the mib mutant showed a significant rescue of the HSC phenotype, implying that angptl2 acts downstream of mib signaling. When MOs against angptls 1 and 2 were then injected into the NICD overexpressing Tg embryos, we found that NICD restored HSC formation in the angptl double morphants. Together, these data corroborate the hypothesis that angptls can regulate notch signaling by acting downstream of notch ligand-receptor interaction and upstream of NICD during definitive hematopoiesis. To further probe at the downstream signaling of angptls since their receptor is currently unknown, we found that both angptls can stimulate activation of akt in cultured endothelial cells. To investigate whether akt is involved in angptl signaling during development, we injected the constitutively active myr-akt into angptl double morphants and observed rescue of both HSC and vascular defects. This strongly indicates that akt is the key signaling component downstream of angptl signaling during these biological processes. Injection of myr-akt can also rescue HSC defects in mib, suggesting that notch signaling also requires akt activation for definitive HSC formation. Taken together, our data demonstrate that the angptls are required for HSC development through notch and akt signaling, by coordinating the production of a functional hemogenic endothelium.