TLR4–MYD88–NF-κB signaling is essential for HSPC emergence. (A) runx1 and rag1 expression was decreased in tlr4bb and myd88 morphants, but increased in ikbaa morphants; thymic epithelial cell marker foxn1 expression was normal in all of these morphants. Black arrowheads mark expression of runx1 in the AGM region at 24 hpf and 36 hpf, rag1 in the thymus at 4 days post fertilization (dpf), and foxn1 in the thymus at 3 dpf. (B) The number of cmyb⁺kdrl⁺ cells in Tg(cmyb:GFP/kdrl:mCherry) embryos was decreased in tlr4bb and myd88 morphants and increased in ikbaa morphants. White arrowheads mark cmyb⁺kdrl⁺ cells in the AGM region at 36 hpf. Right panel, The quantification. Each bar represents the mean ± SEM of 3 independent samples. Each sample was composed of at least 5 embryos. *P < .05, **P < .01. (C) The hematopoietic cells in Tg(CD41:GFP) embryos were reduced in tlr4bb or myd88 morphants and increased in ikbaa morphants. White arrowheads mark the CD41 GFP⁺ cells in the CHT region at 48 hpf. Right panel, The quantification of CD41⁺ cells. Each bar represents the mean ± SEM of 3 independent samples. Each sample was composed of at least 5 embryos. *P < .05, **P < .01. (D) runx1 expression was decreased in JSH-23–treated embryos at 36 hpf. Black arrowheads mark expression of runx1 in the AGM region. (E) The population of cmyb⁺kdrl⁺ cells in Tg(cmyb:GFP/kdrl:mCherry) embryos was decreased in JSH-23–treated embryos. White arrowheads mark cmyb⁺kdrl⁺ cells in the AGM region at 36 hpf. Right panel, Quantification of cmyb⁺kdrl⁺ cells. Data are presented as mean ± SEM of 3 independent samples. Each sample was composed of at least 5 embryos. *P < .05, **P < .01. (F) The cartoon showed the incross between myd88^+/− embryos. Right panels, The decreased expression of runx1 and cmyb expression in myd88^−/− embryos. Black arrowheads mark expression of runx1 in the AGM region at 24 hpf and 36 hpf, and cmyb expression in the CHT region at 48 hpf.