Recent studies have illustrated the significance of some proinflammatory factors in regulating embryonic hemtopoietic stem cells (HSCs) production ,such as TNFα、IFNγ and TLR4.Il6 is a powerful proinflammatory cytokine that plays a pivotal role in the regulation of immunity,hematopoiesis and other physiological processes.Il6-deficient mice leads to impaired survival and self-renewal of HSCs and early progenitors.Whereas Il6 signaling regulates aspects of adult hematopoiesis,little is known about its role in the developmental specification of HSCs.This has led us to explore the impact of loss of Il6 signaling on embryonic HSC emergence by injecting specific antisense morpholinos (MOs) into zebrafish embryos at 1-2 cell stage.
Here we report that MO knockdown of Il6r and Il6 strongly reduced the number of cmyb+HSCs in or near the floor of the dorsal aorta (DA) at 36 hr post fertilization (36hpf) by wholemount in situ hybridization (WISH)compared with their standard MO(Std MO)siblings(Fig.1a).Consistent with the results above,the number of double-positive cmyb+kdrl+ HSCs was reduced about 50% in the floor of the DA in cmyb:GFP;kdrl:mCherry double transgenic embryos at 48 hpf by confocal microscopy when compared to control embryos(Fig. 1b).To distinguish these reductions were due to a defect in the initial specification of HSCs or in their subsequent maintenance,we performed WISH for runx1, both Il6r- and Il6- deficient embryos showed significant reduction in the number of runx1+ HSCs in the aortic floor at 24 and 28hpf.Meanwhile,Il6r and Il6 morphants had sustained reductions in CD41:eGFP+ HSCs colonizing the caudal hematopoietic tissue (CHT) at 72 hpf using transgenic CD41:eGFP animals. Additionally ,expression of rag1 in thymus was completely or nearly absent in Il6r- and Il6-deficient embryos at 96 hpf(Fig. 1c), further surpporting that T-cell defects in these embryos resulted from early HSPC defects. Together, these results demonstrate that Il6 signaling pathway is important both for early specification and subsequent maintenance of HSC fate.
To begin to determine the mechanism by which Il6 signaling regulates embryonic HSCs emergence in vivo,we first tested the impact of loss of Il6 signaling on vasculogenesis.No vascular abnormalities or impaired arterial specification were observed in Il6r- or Il6-deficient embryos at 28hpf when assayed by WISH for kdrl,efnb2a and dlc at the MO doses used in this study,suggesting that the function of Il6 signaling is required specifically during HSC development independently of their role in developing vasculature.Besides,we found that loss of Il6 signaling caused no increased apoptotic endothelial cells within the DA.Primitive hematopoiesis was also unaffected in our study.Lastly,to investigate the connection between Notch and Il6 signaling in HSC specification,we utilized tp1:eGFP;kdrl:mCherry double transgenic embryos and found that the double-positive tp1+kdrl+ Notch signaling-activated HSCs in the ventral floor of the DA had no significant change at 32-36hpf when blocking the IL6 signaling;embryos treated with notch signaling inhibitor DAPT developed fewer cmyb+ HSCs in the ventral floor of DA at 48hpf by WISH than DMSO-treated siblings;however,when the embryos were injected 200pg Il6r mRNA simutaneously, the HSC defect caused by DAPT could be partially rescued (Fig. 1d).In summary,our studies unravel an unexpected developmental role for Il6 signaling and place its action downstream of Notch signaling to control HSC emergence.
Figure 1. a)Representative cmyb WISH images and qualitative phenotype distribution (normal/down,n≥30 embryos/condition)of Il6r,Il6 and std MO-injected embryos at 36 hpf. b) Maximum projections of 48 hpf cmyb:GFP;kdrl:mCherry double-transgenic embryos injected with Std, Il6r, and Il6 MOs. Arrowheads denote cmyb+kdrl+ HSCs along the DA (left figure).Enumeration of cmyb+kdrl+ HSCs shown in(right figure).Error bars represent mean ± SEM of Std (n = 24), Il6r(n = 22), and Il6(n = 24) morphants. c)phenotype distribution(normal/down/absent,n≥30 embryos/condition) of rag1 expression in Il6r,Il6 and std MO-injected embryos at 96 hpf.d)WISH samples and phenotype distribution(high/medium/low,n≥30 embryos/condition)of cmyb expression in DMSO-treated,DAPT-treated,DAPT-treated and Il6r mRNA-injected simutaneously embryos at 48 hpf .
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.