FIGURE SUMMARY
Title

Quality assurance of hematopoietic stem cells by macrophages determines stem cell clonality

Authors
Wattrus, S.J., Smith, M.L., Rodrigues, C.P., Hagedorn, E.J., Kim, J.W., Budnik, B., Zon, L.I.
Source
Full text @ Science
Fig. 1

Fig. 1. Macrophages make intimate interactions with newly formed HSPCs.
(A) Time-lapse live imaging identifies prolonged cell-cell contacts between runx1+23:EGFP+ HSPCs and mpeg1:mCherry+ primitive macrophages that involve the exchange of fluorescent material. (B) About 20 to 30% of HSPCs interact with macrophages in the CHT at any one time from 56 to 106 hpf. Data are means ± SD. (C) High-resolution tracking of individual runx1+23:mCherry+ cells over several hours in the CHT reveals that most HSPCs eventually make sustained contact with macrophages (>5 min). Data are means ± SD. (D) HSPCs frequently complete a cell division shortly after macrophage interactions. About 81% of HSPC divisions occur within 30 min of a macrophage interaction. (E) About 65% of Fucci+ HSPCs in S, G2, and M phases interact with macrophages at any one time, as compared to less than 20% of Fucci– HSPCs. Data are means ± SD. Data were analyzed by unpaired Student’s t test; ****P < 0.0001.
Fig. 2

Fig. 2. Macrophages in the CHT regulate stem cell clonality.
(A) A schematic overview of the Zebrabow-M system: Animals with 15 to 20 insertions of a multicolor fluorescent cassette are crossed to the draculin:CreERT2 line to enable clonal labeling of lateral plate mesoderm lineages. By treating with 4-hydroxytamoxifen (4-OHT) at 24 hpf just after HSC specification, individual stem cell lineages express specific fluorescent hues that can be quantified in the adult marrow. (B) Families of Zebrabow-M;draculin:CreERT2 animals injected with either clodronate liposomes or the irf8 morpholino exhibit reduced numbers of HSC clones in the adult marrow, even when macrophages are not depleted until after emergence from the VDA. Data are means ± SD. Data were analyzed by unpaired Student’s t test; *P < 0.05 and ***P < 0.001. (C) Macrophages (mpeg1:EGFP+) that have interacted with HSPCs (runx1+23:mCherry+) and removed fluorescent material can be harvested by fluorescence-activated cell sorting (FACS). (D) Macrophages that engage HSPCs are marked by lrp1ab and c1qa. The spectral scale reports z-scores. UMAP, uniform manifold approximation and projection.
Fig. 3

Fig. 3. Calreticulin drives HSPC-macrophage interactions to regulate clonality.
(A) Analysis of differentially enriched potential surface proteins from interacting macrophages identifies three paralogs of Calreticulin. (B) Flow cytometry shows that ~30% of runx1+23:mCherry+ HSPCs stain for surface Calreticulin. (C) Morpholino knock-down of calr3a or calr3b significantly reduces the fraction of HSPCs interacting with macrophages at any one time. Data are means ± SD. Data were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test; ns is not significant, *P < 0.05, and ***P < 0.001. MO, morpholino. (D) Calreticulin paralogs without the ER-retention KDEL sequence were fused to EGFP, driven by the HSPC-specific runx1+23 enhancer, and injected into stable runx1+23:mCherry;mpeg1:BFP embryos. Mosaic animals overexpress Calreticulin in a random subset of HSPCs. The arrow indicates an HSPC overexpressing calr3a engaged by a macrophage. (E) HSPCs overexpressing calr, calr3a, or calr3b are more frequently engaged by macrophages compared with non-overexpressing HSPCs in the same embryos. Overexpressing egfp alone has no effect. Data are means ± SD. Data were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test; ns is not significant, *P < 0.05, **P < 0.01, and ****P < 0.0001. (F) Knock-down of calr3a or calr3b reduces the number of HSC clones that contribute to adult hematopoiesis. Data are means ± SD. Data were analyzed by unpaired Student’s t test; *P < 0.05 and ****P < 0.0001.
Fig. 4

Fig. 4. Macrophages buffer HSPC stress and regulate HSPC expansion.
(A) EdU staining of runx1+23:mCherry embryos injected with either the calr3a, calr3b, or irf8 morpholinos identifies a significant reduction in proliferating HSPCs at 3 dpf. Data are means ± SD. Data were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test; **P < 0.01 and ****P < 0.0001. (B and C) scRNA-seq analysis of runx1+23+ FACS-purified cells from irf8 or control morphants reveals a population of stressed HSPCs that persist in the absence of macrophages and a population of cycling cells enriched in the control sample. (D) Embryonic HSPCs marked by surface Calreticulin exhibit higher levels of ROS. Data are means ± SD. Data were analyzed by unpaired Student’s t test; **P < 0.01. MFI, median fluorescence intensity. (E) ROS inhibition with diphenylene iodonium significantly reduces macrophage-HSPC interactions. Data are means ± SD. Data were analyzed by unpaired Student’s t test; *P < 0.05. DMSO, dimethyl sulfoxide. (F) Expression of il1b by heat shock rescues the effect of macrophage depletion on HSPC proliferation. Data are means ± SD. Data were analyzed by one-way ANOVA with Sidak’s multiple comparisons test; *P < 0.05 and ****P < 0.001.
Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Science
Your Input Welcome
We welcome your input and comments. Please use this form to recommend updates to the information in ZFIN. We appreciate as much detail as possible and references as appropriate. We will review your comments promptly.
Please check the highlighted fields and try again.
Thank you for submitting comments. Your input has been emailed to ZFIN curators who may contact you if additional information is required.
Oops. Something went wrong. Please try again later.