Inflammatory Signaling Regulates Embryonic Hematopoietic Stem and Lymphoid Progenitor Cell Formation

Hematopoietic stem cells (HSCs) are defined as cells that can self-renew and provide robust, long-term multilineage engraftment of immunocompromised adults. Only one HSC is sufficient to reconstitute the entire hematopoietic system of an adult mouse, thus blood should theoretically be a simple organ system to generate from embryonic stem cells or induced pluripotent stem cells. Yet, despite two decades of effort, no one has been able to produce HSCs ex vivo from these sources. In the vertebrate embryo, transient waves of hematopoietic cells are produced prior to the formation of HSCs. While the role of erythrocyte production is self-evident, the necessity of myeloid production in the midgestation embryo is less clear. HSCs differentiate from hemogenic endothelium, and in mammalian embryos briefly accumulate in the aorta/gonad/mesonephros (AGM) region within intra-aortic hematopoietic clusters, although the vast majority of cluster cells are not functional HSCs. Approximately 20% of cells in hematopoietic clusters express a Ly6a-GFP transgene (Ly6a encodes Sca-1), and all lymphoid progenitors, pre-HSCs, and HSCs at embryonic day (E)11.5 are found within the Ly6a-GFP⁺ population. Genetic profiling of Ly6a-GFP⁺ and Ly6a-GFP^- hematopoietic cluster cells from the AGM region revealed enrichment in gene ontology (GO) terms associated with innate immunity and inflammatory response in the Ly6a-GFP⁺ population, suggesting inflammatory signaling may regulate the formation of lymphoid progenitors and HSCs. To identify inflammatory cytokines that may be involved in lymphoid progenitor/HSC formation, we performed explant cultures of E9.5 embryos and added inflammatory factors known to induce Sca-1 expression in the adult mouse. Both type I and type II interferons (IFN-α4 and IFN-γ), and to a lesser extent TNFα, induced Ly6a-GFP expression in hematopoietic cells of explanted embryos. IFN-γ receptor 1 (IFN-γR1) was expressed on >80% of Ly6a-GFP⁺ hematopoietic cluster cells in the AGM, and Stat1 was phosphorylated in response to the addition of either IFN-γ or IFN-α4 to AGM explant cultures, indicating that embryonic hematopoietic stem and progenitor cells (HSPCs) are wired to respond to interferon stimulation. Mouse embryos deficient for IFN-g, the IFN-gR1, or the IFN-a4 receptor had decreased numbers of progenitors with lymphoid potential in the AGM region, as measured by limiting dilution culture on OP9 stromal cells, and IFN-gR1 deficient embryos had fewer HSCs, as determined by transplantation into adult mice. The role of IFN-γ signaling in HSC production in the AGM is evolutionarily conserved, as morpholino (MO) knockdown of IFN-g and its receptor likewise reduced HSPC numbers in the dorsal aorta and caudal hematopoietic tissue of zebrafish embryos, and the subsequent population of rag2 expressing lymphoid cells in the thymus. ChIP-Seq demonstrated that interferon regulatory factor 2 (IRF2) occupied genes in human fetal liver CD34⁺ HSPCs, with GO analysis indicating innate immunity and interferon signaling as significantly enriched processes, further demonstrating that interferon signaling is active during vertebrate development. IRF2 is a negative regulator of interferon signaling in the adult, and consistent with this, MO-knockdown of IRF2 in the zebrafish embryo enhanced the production of HSPCs, whereas knockdown of the positive effector IRF1 mimicked the phenotype of IFN-g loss. Myeloid cells have previously been shown to produce IFNs, and primitive F4/80⁺ macrophages were observed in close proximity to hematopoietic cluster cells in the murine AGM region. To determine if the presence of primitive myeloid cells impacted HSPC formation, macrophage specific as well as pan-myeloid knockdown was employed in zebrafish; macrophage depletion by IRF8-MO significantly reduced runx1 expression in the AGM and HSPC production. Significantly, MO-mediated inhibition of PU.1 function, which depletes the majority of myeloid cells, caused an even more striking loss, suggesting IFN-γ is not the only inflammatory mediator, nor primitive macrophages the only myeloid cells, involved in controlling embryonic HSPC production. Together these data demonstrate that myeloid-mediated inflammatory signaling is active in the developing vertebrate embryo, and in the absence of pathogenic challenge function to regulate the production of lymphoid progenitors and definitive HSCs.