Proinflammatory signaling regulates hematopoietic stem cell emergence

Abstract

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 TNF? activates the Notch and NF-?B 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 TNF?, 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.

Figure 1. Tnfa and Tnfr2 are required for HSC generation

(A) Standard control (Std), Tnfr1, Tnfr2, Tnfa, and Tnfr1 and Tnfr2 morphants were examined by WISH for cmyb expression in the aortic floor at 48hpf. White arrowheads denote cmyb⁺ HSPCs. (B) Quantification of cmyb⁺ HSPCs from (A). Each dot represents total cmyb⁺ cells per embryo. The mean ± S.E.M. for each group of embryos is shown in red. (C) cd41:eGFP transgenic embryos were injected with Std, Tnfr1, Tnfr2, and Tnfa MOs and subjected to flow cytometric analysis at 3dpf. Each bar represents the percentage of cd41:GFP⁺ cells in each sample and is the mean ± S.E.M of 3–7 independent samples of 5 embryos each. (D) Maximum projections of 48hpf cmyb:eGFP; kdrl:memCherry double transgenic embryos injected with Std, Tnfr2, and Tnfa MOs. Arrowheads denote cmyb⁺, kdrl⁺ HSCs along the DA. All views: anterior to left. (E) Enumeration of cmyb⁺, kdrl⁺ HSCs shown in (D). Bars represent mean ± S.E.M of Std (n=13), Tnfr2 (n=13), and Tnfa (n=8) morphants. (F) cmyb⁻, kdrl⁺ endothelial cells and cmyb⁻, kdrl⁺ HSCs were isolated from cmyb:GFP; kdrl:mcherry transgenic fish by FACS at 48hpf and examined for expression of tnfr1 and tnfr2. Bars represent means ± S.E.M. of two biological replicates. (G) Confocal tracking of HSC numbers in the floor of the DA from individual cmyb:eGFP; kdrl:mcherry transgenic animals at 36 and 48 hpf following depletion of Tnfr2 or Tnfa compared to standard control morphants. (H) Means ± S.E.M. of cmyb⁺ cell numbers from (G). (I–J) WISH for the T lymphocyte and thymic epithelial markers rag1 (I) and foxn1 (J) (black arrowheads), respectively, in Tnfr2 and Tnfa morphants compared to Std controls at 4 dpf. All views are ventral, with anteriors to left. Numbers represent embryos with displayed phenotype; ns, not significant; *p<0.05, **p<0.01, ***p<0.001. See also Supplementary Figure 1.

Figure 2. Signaling through Tnfr2 regulates HSC development independently of its role in vascular formation

(A) Std, Tnfr2, and Tnfa morphants were interrogated by WISH for the expression of kdrl at 24hpf. (B) cd41:eGFP; gata1a:dsred double transgenic embryos were injected with Std, Tnfr2, and Tnfa MOs and visualized at 3dpf. Arrowheads indicate cd41:GFP⁺ HSPCs in the CHT located between the caudal artery (CA) and caudal vein (CV). Arrows indicate blood flow direction. (C) Expression of the arterial markers efnb2a, dlc, notch1b, and notch3 in Std, Tnfr2, and Tnfa morphants analyzed by WISH at 28hpf. Arrowheads denote the CA. (D) Maximum projections of fli1a:Gal4; UAS:tnfr2; cmyb:eGFP; kdrl:memCherry transgenic embryos at 48hpf. Region shown includes the DA, and arrowheads denote cmyb⁺; kdrl⁺ HSCs. (E) Enumeration of cmyb⁺; kdrl⁺ HSCs shown in (D). Each dot is the number of kdrl⁺; cmyb⁺ cells per embryo. Means ± S.E.M. for each group is shown in red. **p<0.01. All views are lateral, with anteriors to the left. Numbers in panels represent larvae with indicated phenotype. See also Supplementary Figure 2.

Figure 3. Tnfa and Tnfr2 act upstream of Notch signaling during HSC specification

(A) tp1:eGFP; kdrl:mcherry embryos injected with Std, Tnfr2, and Tnfa MOs were visualized at 26hpf. Arrowheads indicate cells in the floor of the DA with active Notch signaling. (B) Enumeration of tp1⁺, kdrl⁺ HSCs from (A). Each dot represents the number of HSCs per embryo, and red lines indicate means ± S.E.M. ***p<0.001. (C) hsp70:Gal4; UAS:NICD-myc embryos injected with Std, Tnfr2, and Tnfa MOs were heat-shocked at 18hpf and WISH for runx1 was performed at 28hpf. Arrowheads denote HSCs along the DA. (D) kdrl:Gal4; UAS:NICD-myc embryos injected with Std, Tnfr2, and Tnfa MOs were analyzed by WISH for runx1 at 28hpf. NICD⁺ larvae were identified using anti-myc-Alexa488 antibody (top panel). Numbers in panels represent the numbers of larvae with indicated phenotype.

Figure 4. Tnfr2 induces jagged1a in endothelial cells, encouraging HSC specification

(A) kdrl:mcherry⁺ cells from dissected trunks of Std or Tnfr2 morphants were purified by FACS at 28hpf for qPCR. Levels of indicated transcripts along x-axis are shown relative to the housekeeping gene ef1a. Bars represent means ± S.E.M. of duplicate samples. (B) AGM regions from fli1a:Gal4; UAS:Tnfr2 embryos were dissected and subjected to qPCR for transcripts shown along x-axis. Bars represent means ± S.E.M. of expression relative to the housekeeping gene rps11. (C) Std (top panels) or Jag1a (bottom panels) morphants were interrogated for runx1 expression at 26hpf and efnb2a and dlc at 28hpf by WISH. Numbers represent larvae with indicated phenotype. (D) Enumeration of runx1⁺ cells in Tnfr2 and/or Jag1a morphants at 28hpf. (E) Enumeration of runx1⁺ cells in Tnfr2 and/or Notch1a and/or Notch1b morphants at 28hpf. Each dot is the number of HSCs per embryo, and red lines indicate means ± S.E.M. (D–E) ***p<0.001; ns, not significant. See also Supplementary Figure 3.

Figure 5. NF-κB is active in emerging HSCs

(A) Trunk region of kdrl:mcherry; NFKB:GFP double transgenic animals visualized by confocal microscopy at 24hpf (left panels) and 30hpf (right panels). Each image is a 2μm z-slice. Arrowheads denote HSCs. (B) cmyb⁻, kdrl⁺ endothelial cells and cmyb⁺, kdrl⁺ HSCs were isolated by FACS at 48hpf. Levels of the NF-κB target genes ikbaa and il1b, as well as the HSC marker cmyb are shown relative to ef1a. Bars represent means ± S.E.M. of two biological replicates. (C) Schematic representation of the experimental design of (D). 28hpf kdrl:mcherry animals were transversally sectioned and subjected to double immunohistochemistry for mcherry (red) and NF-κB (green). DAPI (blue) was added to visualize nuclei. (D) Maximum projections of 1μm sections. Arrowhead indicates a potential HSC emerging in the DA. DA and CV are demarcated by dashed white lines. Yellow asterisks indicate pronephric ducts. (E) tp1:nls-mcherry; NFKB:GFP animals were visualized by confocal microscopy at 24hpf. Each image is a 2μm z-slice. Arrowheads indicate HSCs. CV, caudal vein; DA, dorsal aorta; N, neural tube; Nc, notochord; YE, yolk extension. See also Supplementary Video 1.

Figure 6. NF-κB is required for HSC specification and acts downstream of Tnfr2

(A) hsp70:Gal4; UAS:dn-ikbaa embryos were heat-shocked at 20 hpf. WISH for cmyb was performed at 48hpf. (B) WISH for cmyb in fli1:Gal4; UAS:dn-ikbaa⁻ (left) and fli1:Gal4; UAS:dn-ikbaa⁺ (right) embryos. Arrowheads mark cmyb⁺ cells along the DA. (C) kdrl:mcherry⁺ cells were FACS sorted from Std or Tnfr2 morphants at 28hpf for qPCR. Levels of the NF-κB target gene il1b are shown relative to ef1a. Bars represent means ± S.E.M. from duplicate samples. See also Supplementary Figure 4.

Figure 7. Primitive myeloid cells play a key role in HSC specification

(A) Primitive neutrophils (mpx:GFP⁺) and macrophages (mpeg:GFP⁺) were isolated at 20–24 and 72 hpf by FACS and tnfa expression was quantified by qPCR. Expression was normalized to ef1a and is presented relative to whole embryo expression. Bars represent means ± S.E.M. of two independent experiments. (B) Enumeration of cmyb⁺; kdrl⁺ HSCs shown in (C). (C) Maximum projections of representative images of cmyb:eGFP; kdrl:m Cherry embryos at 48hpf following injections of Std or pU.1 MOs, the latter at two different concentrations. Region shown is the DA, and arrowheads denote cmyb⁺; kdrl⁺ HSCs. (D) Maximum projections of representative images of cmyb:eGFP; kdrl:memCherry embryos at 48hpf in Std and irf8 morphants. Arrowheads denote cmyb⁺; kdrl⁺ HSCs. (E) Enumeration of cmyb⁺; kdrl⁺ HSCs shown in (D). (F) tnfa expression relative to ef1a in 28hpf Std, pU.1 or irf8 morphants. Bars represent means ± S.E.M of duplicate samples. (G) Std, irf8, and irf8+Tnfa morphants were interrogated by WISH for runx1 and mpx at 28hpf. All views are lateral, with anteriors to the left. Numbers represent larvae with indicated phenotypes. *p<0.05; ***p<0.001. See also Supplementary Figure 5.