Estrogens Act As a Dorsal-Ventral Limiting Factor To Pattern The Hematopoietic Stem Cell Niche
Authors: Carroll Kelli J. , Dovey Michael C , Garnaas Maija K , Cutting Claire C , Frechette Gregory M , Esain Virginie , Nissim Sahar , Kwan Wanda , Harris James M , Gorelick Daniel , Halpern Marnie , Goessling Wolfram , North Trista E. 
Published: October 2019

While the genetic control of hematopoietic stem cell (HSC) function is increasingly understood, less is known about factors that pattern the embryonic hematopoietic niche and specify the location of HSC emergence. 17beta-estradiol (E2) and related estrogenic compounds were identified in a zebrafish chemical genetic screen as modifiers of the number of runx1+ HSCs in the Aorta-Gonad-Mesonephros region. Exposure to exogenous E2 during hematopoietic specification significantly decreased production of runx1+ HSCs by in situ hybridization (ISH) and qPCR (p<0.01); this finding was confirmed by CD41 FACS (p<0.001). Morpholino knockdown of the estrogen receptors indicated E2-mediated loss of HSCs was due to Esr2a signaling. Zebrafish that express GFP under the control of the estrogen response element (ERE:GFP) showed endogenous estrogen activity and a 1.5 fold enhancement of GFP expression by FACS upon E2 treatment (p<0.01) confirming that zebrafish embryos contain endogenous estrogen and the transcriptional machinery to respond to exogenous E2; estradiol immunoassay revealed that endogenous estrogen is preferentially located in the yolk of developing embryos. E2 treatment from 12-24 hours post fertilization (hpf), the time in which hemogenic endothelium is specified, disrupted vessel formation as shown by flk1 (kdrl) and altered the assignment of hemogenic arterial identity as assessed by the arterial markers ephrinB2a and tbx20. As arterial specification is mediated by a cascade of sonic hedgehog, VEGF, and Notch, we examined if these pathways were altered. Consistent with a defect in the specification of hemogenic endothelium, E2 decreased expression of VEGFAa and arterial Notch pathway components deltaC and notch3 by ISH and qPCR (p<0.05). Induction of VEGFAa or Notch activity via heat-shock inducible lines rescued E2-mediated hematopoietic defects, suggesting that alterations in these networks underlie observed hematopoietic phenotypes. Further, repression of endogenous estrogen by the aromatase inhibitor anastrozole or the estrogen receptor antagonist fulvestrant increased levels of runx1, the arterial markers ephrinB2a and tbx20, and VEGFAa expression. Expression of venous flt4 was correspondingly decreased, demonstrating that endogenous estrogen signaling regulates arterial/venous identity. These results suggest that maternally deposited estrogens act as a novel morphogen organizer in patterning the HSC niche during early development. It further indicates that endogenous estrogen signaling acts to limit the ventral boundary of hemogenic endothelium by antagonizing somitic VEGFAa, thereby implicating estrogen as a critical regulator of the spatially restricted induction of HSCs. To determine if E2 could impact HSCs independently of its role in specification of hemogenic endothelium, we exposed embryos to E2 from 27-34 hpf, after arterial establishment and initiation of blood flow. Here, E2 enhanced HSCs by ISH and qPCR (runx1; p<0.05). qPCR levels of the cell cycle markers cyclinD1 (p<0.01) and c-myc (p<0.001) and AGM phospho-histone H3 immunoreactivity (p<0.01) increased, suggesting E2 enhances cell cycling. To determine if the ability of E2 to enhance proliferation was restricted to HSCs or was apparent on any blood progenitor population, we assessed the impact of E2 on primitive hematopoiesis. E2 treatment from 12-36 hpf enhanced expression of gata1 by ISH, FACS, and qPCR (p<0.05) while repressing the expression of globin (p<0.05), suggesting that while E2 enhances the self-renewal of progenitors, it impairs their differentiation into mature lineages. To analyze conservation of effect, we examined hematopoietic development in murine embryos with 5-alpha reductase deficiency. These embryos fail to metabolize testosterone into dihydroxytestosterone and as a result have more testosterone available for conversion by aromatase into estradiol, leading to increased estrogen levels. Homozygote pups show fewer phenotypic HSCs by FACS; heterozygotes display increased erythrocyte progenitors but no increase in mature erythrocytes, suggesting that the impact of estrogen on HSC induction and erythropoiesis is conserved in a mammalian system and is sensitive to dose. Together, these data indicate a novel role for estrogen in the regionalization of the hematopoietic niche and identify estrogen as an enhancer of HSC proliferation and maturation.

Disclosures:

No relevant conflicts of interest to declare.