Hematopoietic Stem Cell Formation in Zebrafish Is Regulated in a Temporally Distinct Manner by Estrogen Receptor 2

Abstract 1268

The intrinsic signaling pathways regulating hematopoietic stem cells (HSC) are increasingly understood; in contrast, less is known about the potential effect of exposure to environmental factors, such as xenoestrogens, on the formation of HSCs. RUNX1 (AML1) is a highly conserved transcription factor that is required for definitive HSC induction and is also the target of many chromosomal alterations in leukemia. Through a chemical genetic screen, estrogen-related compounds were identified as modifiers of runx1 expression in the zebrafish. Exposure to 17b-estradiol (E2) throughout hematopoietic development (5 somites (som) to 36 hours post fertilization (hpf)) significantly decreased the number of runx1+ HSCs in the zebrafish Aorta-Gonad-Mesonephros Region (AGM) compared to controls (n≥25–50 embryos /condition). Use of the nonspecific estrogen receptor inhibitor fulvestrant confirmed that estrogen was required for HSC regulation and functioned through classical estrogen receptors. Microarray analysis of FACS-sorted cell populations during zebrafish development demonstrated differential spatio-temporal regulation and expression of esr1 (esrα) and esr2a/b (esrβ) in vascular and hematopoietic cell types; use of an ERE-GFP reporter fish verified that estrogen signaling is active during this stage of embryonic development. 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. Alterations in arterial specification appear to be mediated by the Notch/VEGF pathway. E2 exposure from 5 somites to 36 hpf decreased GFP expression in notch reporter fish as well as expression of deltaC and notch5 by in situ hybridization. Interestingly, when exposure to E2 or DPN occurred from 24 – 36 hpf, after arterial establishment and 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 acridine orange) and pro-proliferative (BrdU+) effects on HSCs and the vascular niche depending on the timing of exposure. 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, modifications in Wnt activity were seen post-E2 exposure from 24 – 36 hpf. To determine whether 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 that all decreased formation of HSCs; using fulvestrant and the ERE-GFP reporter, we confirmed that the phenotype elicited by each was at least partially 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.