The intrinsic signaling pathways regulating hematopoietic stem cells (HSC) are increasingly well recognized. However, less is known about how in utero exposure to common environmental xenobiotic compounds may alter HSC development and increase the risk of carcinogenesis. RUNX1 (AML1), required for definitive HSC induction in all vertebrates, is the target of frequent chromosomal alterations associated with leukemia. Through a chemical genetic screen for modifiers of runx1 expression in the zebrafish, estrogen-related compounds were identified. Here, we found that exposure to 17β-estradiol (E2) throughout the initial waves of hematopoietic development (5 somites (som) to 36 hours post fertilization (hpf)) significantly altered the number of runx1+ HSCs in the zebrafish Aorta-Gonad-Mesonephros Region (AGM) compared to controls (n≥25-50 embryos /condition). Other physiological estrogens, such as estrone and estriol, elicited a similar hematopoietic response. However, treatment with either the isomer 17α-estradiol, or the related steroid hormones testosterone or progesterone, could not mimic the effect of E2 on HSCs. Use of the aromatase inhibitor anastrozole and the pan-estrogen receptor inhibitor fulvestrant confirmed that estrogen was both required for nascent HSC regulation and functioned through classical estrogen receptor (esr) signaling. Microarray analysis of FACS-sorted cell populations during zebrafish development demonstrated differential spatio-temporal regulation of esr1 (esrα) and esr2a/b (esrβ) in vascular and hematopoietic cell types. During the primitive wave of hematopoiesis, exposure to E2 and the esr1-agonist PPT significantly enhanced red blood cell number as seen by in situ hybridization for embryonic globin (hbbe3) and quantified by fluorescent microscopy and FACS analysis of the Tg(globin:GFP) line. Conversely, the esr2-specific agonist DPN diminished definitive HSC formation after exposure from 5 som to 24 hpf; this phenotype was mediated by disruption of vessel formation, as indicated by flk1 (kdrl) expression, and alteration in the assignment of artery-vein identity. Interestingly, when exposure to E2 or DPN occurred from 24 – 36 hpf, after the establishment of ephb2+ arteries and the initiation of blood flow, estrogen treatment enhanced HSC formation; this was confirmed by FACS analysis and fluorescent microscopy using the Tg(cmyb:eGFP) and Tg(-6.0itga2b:eGFP)la2 (CD41:GFP) HSC-reporter lines. E2 treatment was found to elicit both pro-apoptotic (TUNEL+) and pro-proliferative (BrdU+) effects on HSCs and the vascular niche depending on the timing of exposure, but independent of the concentration of E2 over the physiological range and above (10nM to 10mM). Morpholino-mediated gene knockdown of esr1 and the two esr2 alleles alone and in combination with E2 confirmed that esr2 was responsible for the effects on definitive hematopoiesis. Using the Tg(TOP:GFP)w25 line, alterations in estrogen signaling were shown to mediate effects on wnt activity. To determine whether exposure to environmental estrogens could mediate similar alterations in HSC specification and proliferation, we exposed embryos to the phytoestrogen genistein, the synthetic estrogen ethinylestradiol, and the xenoestrogen bisphenol A (BPA) and found results reminiscent of E2; using fulvestrant, we confirmed that the phenotype elicited by each was dependent on estrogen receptor stimulation. In an adult zebrafish marrow injury model, E2 significantly enhanced stem and progenitor cell regeneration in males and females by day 10 post irradiation (n≥10 /condition). Intriguingly, we found that females, with higher circulating estrogen levels, recovered better after injury than male siblings, both in the presence and absence of exogenous estrogen. Finally, murine bone marrow treated with E2 or DPN produced significantly (n=10 /condition, p<0.0001) higher numbers of spleen colonies at day 12 post-transplantation than vehicle-only controls, demonstrating functional conservation of estrogenic regulation of HSCs/progenitor cells. These data identify stage-specific, differential roles for estrogen during hematopoiesis, highlighting the potent impact of environmental exposure to estrogenic compounds on blood formation and revealing potential therapeutic options for the treatment of bone marrow failure and leukemia. (equal contribution: KJC, MCD; WG, TEN).
Goessling:Fate Therapeutics: Consultancy, Patents & Royalties. North:Fate Therapeutics: Consultancy, Patents & Royalties.
Asterisk with author names denotes non-ASH members.