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Fig. 2 Tlr3 is required for HSPC grooming. (A) Stack Plot showing the macrophage interaction behavior in the presence of the Calr inducers. Grooming behavior is shown in the black bar, and dooming behavior as the orange bar. The imaging was conducted using 3-dpf zebrafish embryos. The exact sample size is indicated in the figure. n = 6 to 16 from two independent experiments. Data were analyzed by Wilcoxon matched-pairs signed rank test. *P = 0.0156, **P = 0.002, ***P = 0.001. (B) Schematic overview of the Gecko 2.0 CRISPR-Cas9 knockout screen. (C) sgRNAs significantly enriched (P < 0.05) were identified using MAGECK software and plotted as a cumulative stack on the basis of their Z score. The arrows indicate examples of significantly enriched sgRNAs, and their Z score is depicted inside the brackets. The screen was conducted using K562 cells. The screen was performed in three independent experiments. (D) Knock-down of Tlr3 (Tlr3 MO) or iTlr3 treatment reduces the fraction of HSPCs that are groomed but increases the fraction of HSPCs that are doomed in 3-dpf zebrafish embryos. The macrophage behavior outcome fraction was calculated by counting the total number of interacting macrophages and dividing by the total number of dooming or grooming events. Data points represent an image embryo: SD MO, n = 9; tlr3 MO, n = 11; DMSO, n = 4; and iTLR3, n = 5. Two independent experiments were performed. Two-way ANOVA followed by Sidak?s multiple comparisons test was used. **P = 0.0027; ***P = 0.00005. (E) Representative imaging showing the experimental design of the parabiosis experiment. (F) Both macrophages showed higher interaction rates with the HSPCs. *P = 0.04, **P = 0.0015. Data were analyzed using a two-way ANOVA test. The macrophage-HSPC interaction percentage was calculated by counting the total number of HSPCs (runx1+ cells) and dividing by the total number of HSPCs interacting with a macrophage. (G) Bar plot showing the macrophage behavior outcome fraction. The macrophage behavior percentage outcome was calculated by counting the total number of interacting macrophages and dividing by the total number of dooming or grooming events. *P = 0.01, **P = 0.0057. Data were analyzed using an unpaired Student?s t test. For (F) and (G), data points represent an image embryo. n = 10 from three independent experiments.