Fig 2. Hematopoietic-specific loss of Zeb1 leads to differentiation defects in specific HSPC populations as well as myeloid lineage and T-cell defects.
(A) Schematic of BM transplant experiments using Zeb1 null CD45.2+ fetal liver HSPCs from Tie2-Cre, Zeb1fl/fl mice, and Cre only controls transplanted into CD45.1+ recipients with PB and BM analysis conducted at 23 weeks post-transplant. (B) Overview of flow cytometry gating strategy used to analyze hematopoietic stem and progenitor (HSPC) populations. LSK cells were analyzed for SLAM marker expression (CD150, CD48) or were analyzed by parallel (CD135/CD34) marker expression to define MPP as well as ST and LT HSC populations more accurately. MPPs were analyzed by FcgammaR, CD34 expression to further define MEP, GMP, and CMP populations. (C) SLAM marker expression showing similar percentage of LT-HSCs (CD150+CD48−) in Zeb1 null and control BM. (D) Overall, there were decreases in the percentage of Lin−Sca+cKit+ cells likely composed of significant decreases in the percentage Zeb1-deficient BM percentages for ST-HSCs and MPPs. Moreover, there were significant decreases in the percentage of as CMP and GMP cell populations in the BM of Zeb1 null reconstituted recipients. (E) Flow cytometric analysis of PB (left panel), BM of reconstituted mice showed defects in Zeb1 null HSPC contribution to myeloid cells (Cd11b+) including monocytic (Cd11b+Ly6G) and NEU (Cd11b+Ly6G+) lineage cells. (F) Cytometric analysis of thymic T-cell populations showed significantly decreased percentage of CD25−CD44− DN4 progenitors and increased CD8+ mature T cells. Error bars indicate SD of the mean (n = 4 per group, *p < 0.05, **p < 0.01, ****p < 0.0001, nonparametric t test). Raw data behind graphs are included in A in S1 Data. BM, bone marrow; HSPC, hematopoietic stem and progenitor cell; LSK, Lin−Sca1+cKit+; LT-HSC, long-term HSC; MPP, multipotent progenitor; NEU, neutrophil; PB, peripheral blood; ST-HSC, short-term HSC.