Extended Data Fig. 7: Restoring Notch1 signaling in Fli-1 deficient HSPCs rescues their expansion capacities.
From: Transcriptional activation of regenerative hematopoiesis via microenvironmental sensing
a, Frequency of Notch1+ BM LSK and SLAM LSK HSPC from WT and Fli-1ROSAΔ chimeric mice (described in Extended Data Figs. 2b), 7 days post tamoxifen induction. Unpaired two tailed t-test was used; n = 5 per WT or Fli-1ROSAΔ. Data are presented as mean values +/- SEM. b, c, WT and Fli-1ROSAΔ BM HSPCs were isolated and expanded. b, Flow cytometry determined frequency of Vegfa expressing LSK HSPCs. Unpaired two tailed t-test was used; n = 5 tissue donor mice per genotype, each point represents an average of 3-4 technical replicates. Data are presented as mean values +/- SEM. c, Representative Vegfa histogram plots gated from LSK HSPCs. Histogram plot bars indicate the gates for the positive fluorescent signal. d, Frequency of Vegfa+ BM LSK and SLAM LSK HSPC from WT and Fli-1ROSAΔ chimeric mice (described in Extended Data Figs. 1b), 7 days post tamoxifen induction. Unpaired two tailed t-test was used; n = 5 per WT or Fli-1ROSAΔ. Data are presented as mean values +/- SEM. e, IGV peaks plot for the genomic loci of Vegfa, displaying bigWig coverage tracks for Fli1-ChIP-seq and Spi1-ChIP-seq. f, Representative flow dot plots for WT, Fli-1ROSAΔ, Fli-1ROSAΔN1-ICiOE, and N1-ICiOE HSPCs post expansion in co-culture with a vascular niche, gated from CD45+/Lin- cells. g, Frequency of hematopoietic cells was determined by flow cytometry, number of cells was determined out of total MNC counts, and fold expansion was calculated. One-way ANOVA multiple comparisons was used; n = 4 BM donor mice per genotype, with 2 technical replicates per donor. Data are presented as mean values +/- SEM. h, HSPC frequency as determined by flow cytometry. One-way ANOVA multiple comparisons was used; n = 4 BM donor mice per genotype, with 2 technical replicates per donor. Data are presented as mean values +/- SEM. i, Proposed model for Fli-1 endorsed HSPC activation via Notch/Vegfa pathways mediated crosstalk with the vascular niche. Fli-1 in HSPCs, transcriptionally presets Notch pathway-promoting external and internal elements while also transcriptionally promoting Vegfa expression. Vegfa enlists an activation supportive vascular niche by stimulating ECs to elevate the expression of Notch ligands. Reciprocal niche-mediated activation of Notch receptors presented by HSPCs, endorse downstream Notch signaling, promoting HSPC regenerative activation and expansion. Created with BioRender.com.
