Summary Hematopoietic stem cells (HSCs) underlie the production of blood and immune cells for the lifetime of an organism. In vertebrate embryos, HSCs arise from the unique transdifferentiation of hemogenic endothelium comprising the floor of the dorsal aorta during a brief developmental window. To date, this process has not been replicated in vitro from pluripotent precursors, partly because the full complement of required signaling inputs remains to be determined. Here, we show that TNFR2 via TNFα activates the Notch and NF-κB signaling pathways to establish HSC fate, indicating a requirement for inflammatory signaling in HSC generation. We determine that primitive neutrophils are the major source of TNFα, assigning a role for transient innate immune cells in establishing the HSC program. These results demonstrate that proinflammatory signaling, in the absence of infection, is utilized by the developing embryo to generate the lineal precursors of the adult hematopoietic system.

It has long been established that lower vertebrates, most notably fish and amphibians, are resistant to the toxic effect of LPS. Furthermore, the lack of a TLR4 ortholog in some fish species and the lack of the essential costimulatory molecules for LPS activation via TLR4 (i.e., myeloid differentiation protein 2 (MD-2) and CD14) in all the fish genomes and expressed sequence tag databases available led us to hypothesize that the mechanism of LPS recognition in fish may be different from that of mammals. To shed light on the role of fish TLRs in LPS recognition, a dual-luciferase reporter assay to study NF-κB activation in whole zebrafish embryos was developed and three different bony fish models were studied: 1) the gilthead seabream (Sparus aurata, Perciformes), an immunological-tractable teleost model in which the presence of a TLR4 ortholog is unknown; 2) the spotted green pufferfish (Tetraodon nigroviridis, Tetraodontiformes), which lacks a TLR4 ortholog; and 3) the zebrafish (Danio rerio, Cypriniformes), which possesses two TLR4 orthologs. Our results show that LPS signaled via a TLR4- and MyD88-independent manner in fish, and, surprisingly, that the zebrafish TLR4 orthologs negatively regulated the MyD88-dependent signaling pathway. We think that the identification of TLR4 as a negative regulator of TLR signaling in the zebrafish, together with the absence of this receptor in most fish species, explains the resistance of fish to endotoxic shock and supports the idea that the TLR4 receptor complex for LPS recognition arose after the divergence of fish and tetrapods.

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How fish larvae are protected from infection before the maturation of adaptive immunity, a process which may take up to several weeks in most species, has long been a matter of speculation. Using a germfree model, we show that colonization by commensals in newly hatched zebrafish primes neutrophils and induces several genes encoding proinflammatory and antiviral mediators, increasing the resistance of larvae to viral infection. Commensal microbe recognition was found to be mediated mainly through a TLR/MyD88 signaling pathway, and professional phagocytes were identified as the source of these immune mediators. However, the induction of proinflammatory and antiviral genes, but not of antimicrobial effector genes, also required the covalent modification of histone H3 at gene promoters. Interestingly, chromatin modifications were not altered by commensal microbes or hatching. Taken together, our results demonstrate that gene-specific chromatin modifications are associated with the protection of zebrafish larvae against infectious agents before adaptive immunity has developed and prevent pathologies associated with excessive inflammation during development.epigenetic | cytokines | evolution | gene regulation | live imaging