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Inflammatory signaling regulates hematopoietic stem and progenitor cell emergence in vertebrates

Qiuping He, Chunxia Zhang, Lu Wang, Panpan Zhang, Dongyuan Ma, Junhua Lv and Feng Liu

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Article Figures & Data

Figures

  • Figure 1

    Inflammatory signaling is enriched in hemogenic endothelium. (A) runx1 GFP+ cells from Tg(runx1:en-GFP) transgenic embryos at 26 to 28 hpf were sorted for deep sequencing. The GO analysis of the sequencing data identified that inflammatory signaling was enriched in runx1+ cells. (B) Heat map analysis showed comparison of gene expression between runx1 GFP+ and runx1 GFP cells at 26 to 28 hpf. Multiple inflammatory signaling genes were found enriched in GFP+ cells. (C) cmybkdrl+ and cmyb+kdrl+ cells from Tg(cmyb:GFP/kdrl:mCherry) embryos were sorted at 26 to 28 hpf, and lyz+ cells from Tg(lyz:DsRed) embryos were sorted at 48 hpf. (D) qRT-PCR results showed that a panel of inflammatory signaling genes was enriched in cmyb+kdrl+ hemogenic endothelial cells. Each bar represents the mean ± SEM of 3 independent samples. *P < .05, **P < .01.

  • Figure 2

    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.

  • Figure 3

    NF-κB derived from endothelial cells mediates the TLR regulation of HSPC emergence. (A) Top panel, Endothelial dnIκBaa-GFP+ signals. Red arrowheads mark GFP signal in the endothelial cells. Bottom panels, The decreased expression of p-IKBa in dnikbaa and dnikbaa+ embryos. (B-C) Overexpression of dnikbaa in endothelial cells led to decreased expression of runx1. (D) Decreased expression of runx1 at 36 hpf in tlr4bb or myd88 morphants was rescued by coinjection of ikbaa MO. (E) The decreased population of cmyb+kdrl+ cells in Tg(cmyb:GFP/kdrl:mCherry) embryos injected with tlr4bb or myd88 MO, was rescued by coinjection with ikbaa MO. White arrowheads mark cmyb+kdrl+ cells in the AGM region at 36 hpf. (F) 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. (G) Decreased expression of runx1 at 36 hpf in tlr4bb or myd88 morphants was rescued by the overexpression of il1b from 20 hpf.

  • Figure 4

    Notch signaling functions downstream of inflammatory signaling to regulate HSPC emergence. (A) qRT-PCR results from the dissected trunk region showed that expression of Notch target genes hey1, hey2, and her15.1 was decreased in tlr4bb and myd88 morphants and increased in ikbaa morphants at 28 hpf. Each bar represents the mean ± SEM of 3 independent samples. *P < .05, **P < .01. (B) hey2 expression was decreased in myd88−/− embryos. Black arrowheads mark expression of hey2 in the dorsal aorta at 24 hpf. (C) tp1+fli1a+ cells in Tg(tp1:DsRed/fli1a:EGFP) transgenic embryos were decreased in tlr4bb or myd88 morphants and increased in ikbaa morphants. Left panels, Confocal images of the tp1+fli1a+ cells in the AGM region at 36 hpf, which were labeled by the white arrowheads. Right panel, Quantification of the tp1+fli1a+ cells. Each bar represents the mean ± SEM of 3 independent samples. Each sample was composed of at least 6 embryos. *P < .05, **P < .01. (D) Overexpression of NICD in endothelial cells partially rescued the HSPC defect in tlr4bb or myd88 morphants. Top panel, The NICD-GFP+ signals. Bottom panels, The expression of runx1 at 36 hpf in the embryos coinjected with tlr4bb or myd88 MOs and fli1a-ep-NICD-GFP construct. Red arrowheads mark GFP signal in the endothelial cells. Black arrowheads mark the expression of runx1 in the AGM region at 36 hpf.

  • Figure 5

    The deficiency of inflammatory receptors leads to HSPC defects. (A) Expression of HSPC marker runx1 was decreased in mcsfr, gcsfr, and tnfr2 morphants at 24 hpf. Black arrowheads labeled expression of runx1 in the AGM region. (B) Fluorescence-labeled hemogenic endothelial cells were decreased in Tg(cmyb:GFP/kdrl:mCherry) embryos injected with gcsfr or tnfr2 MO at 36 hpf. White arrowheads mark cmyb+kdrl+ cells in the AGM region. Each bar represents the mean ± SEM of 3 independent samples. Each sample was composed of at least 5 embryos. **P < .01. (C) qRT-PCR analysis of the NF-κB target gene il1b showed that the decreased expression of il1b in gcsfr and tnfr2 morphants at 36 hpf were rescued by coinjection of ikbaa MO. Each bar represents the mean ± SEM of 3 independent samples. *P < .05, **P < .01. (D) The decreased expression of HSPC marker runx1 in gcsfr and tnfr2 morphants were rescued by coinjection of ikbaa MO. Black arrowheads mark runx1 expression in the AGM region at 36 hpf.

  • Figure 6

    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.