Inflammatory signaling plays an evolutionarily conserved role in mouse definitive hematopoiesis. (A) Immunofluorescence of Runx1 in the E10.5 AGM region of tlr4^+/− and tlr4^−/− embryos. Right panel, The quantification of Runx1⁺ cells in AGM region on the sections (tlr4^+/−, n = 3; tlr4^−/−, n = 3). (B-D) qRT-PCR analysis showed attenuated expression of HSC markers (B), NF-κB signaling target genes (C), and Notch target genes (D), respectively. (E) CFC assay of AGM regions showed the numbers of CFU-Mix, CFU-GM, and BFU-E were decreased in tlr4^−/− embryos. One embryo equivalent was used. (F) The scheme of cell sorting and HSC transplantation assay in mouse embryos. (G) Flow cytometry results showed an increased amount of C-Kit⁺CD45⁺ cells in tlr4^−/− embryos after 4 days of OP9 coculture. (H) Donor-derived chimerism in recipients. Symbols represent the donor chimerism in bone marrow of each recipient at 2 months posttransplantation. (I) The donor-derived multilineage reconstitution was shown by the presence of CD45.2 cells in the myeloid (Gr1⁺/CD11b⁺), B lymphoid (B220⁺), and T lymphoid (CD3⁺, or CD4⁺/CD8⁺) populations of bone marrow, spleen, and thymus in a representative recipient at 2 months posttransplantation. (J) Model of inflammatory signaling during HSPC emergence. The inflammatory signals were required to activate NF-κB–Notch signaling in the hemogenic endothelial cells, and then regulate HSPC development. (B-E) Each bar represents the mean ± SEM of 3 independent samples. *P < .05, **P < .01.