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Fig. 3

ID

ZDB-FIG-220923-20

Publication

Wattrus et al., 2022 - Quality assurance of hematopoietic stem cells by macrophages determines stem cell clonality

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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.

Expression Data

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Antibody Labeling

Phenotype Data

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Acknowledgments

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