Fig 1.
Expression analysis of Zeb1 and Zeb2 during early hematopoiesis.
(A) Levels of Zeb1 (upper) and Zeb2 (lower) across LT-HSC (dark green to light green channels, Lin−c-Kit+ Sca1+CD34–Flk2–), HSPC (dark red to purple, LSK) and progenitors (dark red to light red channels, LSK), respectively. Zeb2 levels are higher throughout except for ST-HSCs where Zeb1 levels are higher (asterisk). Heatmaps at the bottom of graphs show percentage of cell lineage that express Zeb1 or Zeb2. HSPC subpopulations were subdivided into HSC1 and HSC2 (projected) expressing low/absent and high SLAM marker CD229, respectively, including ESLAM cells (CD45+EPCR+CD48−CD150+). (B) (Upper) dimensionality reduction and trajectories of 3 major cell types (LT-HSC, HSPC, and Prog) across 1,920 hematopoietic single cells sequenced by Nestorowa and colleagues [19], analyzed with the Monocle3 algorithm [20]. Clusters identified by Monocle3 were enclosed in a black box. Two major clusters consisting in LT-HSC and HSPC (see clusters 1 and 3, respectively) and 1 cluster of progenitors (see cluster 2) were detected. Also, another restricted cluster of LT-HSC were found (see cluster 4). (Lower) Pseudotime fitting analysis of these cells with tradeSeq program [21]. (C) (Left) Expression of Zeb1 gene across the single cell experiment, color coded according to the log10 of Zeb1 pseudocounts. Cells with zero pseudocounts for Zeb1 expression are colored in gray. (Right) Estimated expression obtained for Zeb1 across cell types expressing Zeb1 and arranged by pseudotime with the tradeSeq program. Across pseudotime, fitted curves indicates up-regulation of Zeb1 expression in LT-HSC (black curve), down-regulation of Zeb1 in HSPC (green curve) and mild down-regulation of Zeb1 in progenitors (yellow curve). (D) (Left) Same as left C for Zeb2 gene. (Right) Same as right C for Zeb2 gene. Across pseudotime, fitted curves indicate up-regulation of Zeb2 expression in LT-HSC and HSPC (black and green lines, respectively) and up-regulation of Zeb2 in progenitors (yellow curve). HSPC, hematopoietic stem and progenitor cell; LSK, Lin−Sca1+cKit+; LT-HSC, long-term HSC; ST-HSC, short-term HSC.
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.
Fig 3.
Hematopoietic-restricted loss of Zeb1 decreases hematopoietic colony formation potential and ability to compete with wt HSPCs for contribution to all hematopoietic lineages.
(A) HSPCs isolated from Vav-iCre; Zeb1fl/fl fetal livers show decreased numbers of colonies in methylcellulose-based colony assays (first) that further decreases upon secondary replating (second) compared to Cre negative controls. (B) Schematic of competitive BM reconstitution experiments whereby equal numbers of BM cells from Zeb1 null CD45.2+ mice were mixed with equal numbers of control CD45.1+ cells and used to reconstitute lethally irradiated CD45.1+ mice. If Zeb1 null CD45.2+ HSPCs are not compromised, they would be expected to contribute equally as the control CD45.1+ cells in their contribution to all hematopoietic cells (equal CD45.2+-orange/CD45.1+-blue, top row) whereas if they are severely compromised then the control CD45.1+ cells will solely contribute to the reconstituted hematopoietic system (all CD45.1+- blue, lower row). (C) Zeb1 null (Vav-iCre, Zeb1fl/fl) CD45.2+ donor cells (orange bars) were outcompeted by control CD45.1+ competitor HSPCs (blue bars) for their ability to contribute to all hematopoietic cells analyzed in the PB and BM (rightmost panels). Zeb1 fl/+ heterozygous (middle panels) and Cre negative (left panels) CD45.2+ doner cells in general contributed equally as well as the competitor CD45.1+ cells for their contribution to the hematopoietic system of recipient mice with the exception to the T-cell lineage. Data are represented as mean + SD from 4 biological replicates. *p < 0.05; **p < 0.01, nonparametric t test. Raw data for (A) are included in S1 Data. Raw data for (C) are included in B of S1 Table. BM, bone marrow; HSPC, hematopoietic stem and progenitor cell; PB, peripheral blood; wt, wild-type.
Fig 4.
Double deletion of Zeb1 and Zeb2 causes PB cytopenia and severe differentiation defects in HSPCs.
(A) Schematic of experiments (left panel) used to study the effect of tamoxifen-inducible deletion of Zeb1, Zeb2, or both after donor BM reconstitution (CD45.2+) of lethally irradiated recipients (CD45.1+). Schematic of loxP flanked (floxed-Fl) conditional Zeb1 and Zeb2 alleles before tamoxifen induced Cre-mediated deletion and recombined delta Zeb1 and Zeb2 alleles after recombination (right panel). (B) HCT analysis of BM 10 days after tamoxifen treatment showing decreased HGB (top left) in Zeb1Δ/Δ, Zeb2Δ/Δ, and Zeb1/2Δ/Δ DKO settings. There was as well decreased RBC (top middle), PLT (top right), and GRA (bottom left). LYM (bottom middle) in the BM were decreased only in the Zeb1/2Δ/Δ DKO. (C) Flow cytometric analysis of HSPCs in the BM 10 days after tamoxifen treatment showing increased numbers of LSK cells and well as LT-HSCs (LTS- lin−cKit+Sca1+CD34−Cd125−) in Zeb1/2Δ/Δ DKO settings. (D) Representative flow cytometry analysis for hematopoietic stem and progenitor populations showing increased numbers of LSK and LT-HSCs in Zeb1/2Δ/Δ DKO settings. Bars in panels represent mean ± SD, n = 5 per group; *p < 0.05; **p < 0.01; ****p < 0.0001, Dunnett multiple comparisons test. Raw data behind graphs are included in C of S1 Data. BM, bone marrow; DKO, double knockout; GRA, granulocyte; HCT, hematocrit; HGB, hemoglobin; HSPC, hematopoietic stem and progenitor cell; LSK, Lin−Sca1+cKit+; LT-HSC, long-term HSC; LTS, long-term HSC; LYM, lymphocyte; MPP, multipotent progenitor; PB, peripheral blood; PLT, platelet; RBC, red blood cell; STS, short-term HSC.
Fig 5.
Maintenance of a single Zeb2 wt allele rescues PB cytopenia and severe differentiation defects in Zeb1/2 DKO HSPCs.
(A) Hemavet analysis showing that maintenance of a single wt Zeb2 allele in Zeb2Δ/+, Zeb1Δ/Δ (abbreviated Zeb2Δ/+) mice can rescue the HCT defects observed in Zeb1/2Δ/Δ DKO including RBC and HB levels as well as normalization of PLT (right) numbers. (B) Flow cytometric analysis of HSPCs from the BM showing that presence of single Zeb2 allele in Zeb2Δ/+ mice can also normalize LSK, LT-HSC (lin−cKit+Sca1+CD34−Cd135−), ST-HSC (lin−cKit+Sca1+Cd34+Cd135−), HPC (lin−cKit+Sca1−), CMP, GMP, and MEP HSPC numbers compared to defects observed in Zeb1/2Δ/Δ DKOs. (C) Representative flow cytometry plot of HSPCs. Bars in panels represent mean ± SD, n = 5 per group; *p < 0.05; **p < 0.01; ****p < 0.0001, Dunnett multiple comparisons test. Raw data behind graphs are included in D of S1 Data. DKO, double knockout; HB, hemoglobin; HCT, hematocrit; HSPC, hematopoietic stem and progenitor cell; PB, peripheral blood; PLT, platelet; RBC, red blood cell; wt, wild-type.
Fig 6.
RNA-seq analysis of LSK-enriched populations reveals both common and unique immediate early gene expression programs controlled by Zeb1 and Zeb2.
(A) DEG lists obtained with edgeRun R package, when comparing R26-Cre-ERT2; Zeb2fl/fl (Zeb2Δ/Δ), R26-Cre-ERT2; Zeb2fl/+, Zeb1fl/fl (Zeb2Δ/+, Zeb1Δ/Δ), and Zeb1fl/fl (Zeb1Δ/Δ) against iDKO R26-Cre-ERT2 Zeb1/2 fl/fl (Zeb1/2Δ/Δ), respectively. To define DEGs, we used as cutoff an FDR <0.1. (B) Heatmap of >360 combined ZEB1 and ZEB2 DEGs, sorted from most induced to repressed ZEB2-DEGs. From left to right, we plotted the Z-scores of gene expression of R26-Cre-ERT2 Zeb2 fl/fl (Zeb2Δ/Δ), single R26-Cre-ERT2; Zeb2fl/+ allele, Zeb1fl/fl (Zeb2Δ/+, Zeb1Δ/Δ) and the iDKO R26-Cre-ERT2; Zeb1/2fl/fl (Zeb1/2Δ/Δ), respectively. (C) (Left) Intersections of DEGs from Zeb1 null cells expressing double or a single Zeb2 allele against iDKO cells, respectively. (Right) Intersections of DEGs from Zeb1 or Zeb2 null cells against iDKO cells, respectively. Raw data behind (B) are included in S2 Table. DEG, differentially expressed gene; DKO, double knockout; FDR, false discovery rate; iDKO, inducible double knockout; LSK, Lin−Sca1+cKit+; RNA-seq, RNA sequencing.
Fig 7.
RNA-seq analysis of LSK-enriched populations reveals both common and unique immediate early gene expression programs controlled by ZEB1 and ZEB2.
(A) Associated GO terms with ZEB2-DEGs, obtained with the STRING database, using an FDR <0.05). (B) Associated network analysis for ZEB2-DEGs, obtained with STRING database using highest confidence interaction scores (0.900) and clustered with an MCL inflation parameter of 3. (C) Same as (A) for ZEB1 DEGs. (D) Same as (B) for Zeb1 DEGs. Raw data behind panels are included in S3 Table. DEG, differentially expressed gene; FDR, false discovery rate; GO, gene ontology; LSK, Lin−Sca1+cKit+; RNA-seq, RNA sequencing.
Fig 8.
Zeb1 overexpression leads to extramedullary hematopoiesis/splenomegaly, enhanced myeloid cell development, and monocyte lineage skewing.
(A) Schematic of conditional Rosa26-Zeb1-IRES-EGFP-pA+ transgenic locus (left). (B) Following Vav-iCre-mediated deletion of the loxP flanked transcriptional stop cassette Zeb1 expression is increased approximately 4.5-fold in transgenic BM HSPCs compared to controls (N = three 5-month-old female mice/genotype, p = 0.0125; error bars indicate SD of the mean, Mann–Whitney test) along with (C) dosage-dependent EGFP expression in both heterozygous and homozygous Zeb1 transgenic HSPCs (Flow cytometry for EGFP). (D) Western blot confirmation of increased ZEB1 protein in the BM and spleen of Vav-iCre; Zeb1tg/tg mice compared to Cre-negative control samples. (E) Increased spleen size/extramedullary hematopoiesis seen in Zeb1tg/tg transgenic mice (left panel) showing roughly doubling in size compared to body weight (right panel). (F) Flow cytometric analysis showing increased myeloid cells in spleen (CD11b+, Gr1+). (G) Representative flow cytometry plot showing increased CD11b+, Lys6G- monocytes in the BM of Zeb1tg/tg mice. (H) Increases in myeloid cells (CD11b+, Gr1+) with monocytic skewing/expansion was present in both Vav-iCre (left) and Tie2-Cre models (right). Data are represented as mean + SD from 3 biological replicates/genotype. *p < 0.05; **p < 0.01, nonparametric t test. Raw data behind graphs and western blot in (D) are included in E of S1 and S2 Data, respectively. BM, bone marrow; HSPC, hematopoietic stem and progenitor cell; SP, spleen.
Fig 9.
Inducible deletion of Zeb1/2 increases in vivo survival in MLL-AF9 secondary transplant settings.
(A) Schematic of inducible deletion strategy to investigate the effects of Zeb2 and Zeb1/2 deletion on secondary leukemia progression. (B) Tam-induced Zeb2 deletion was found to significantly increase overall survival of mice transplanted with MLL-AF9 secondary tumor cells compared to nontreated Veh treated controls (median survival 66 versus 42 days, p = 0.0082, Mantel–Cox test). There was a significant effect of Tam in Cre only treated samples compared to Veh controls (median survival 29 versus 19 days, p = 0.0082, Mantel–Cox test). (C) Tam-induced deletion of both Zeb1 and Zeb2 was also found to significantly increase overall survival of mice transplanted with MLL-AF9 secondary tumor cells compared to nontreated Veh treated controls (median survival 58 versus 31 days, p = 0.0031, Mantel–Cox test). There was a significant effect of Tam in Cre only treated samples compared to Veh controls (median survival 42 versus 31 days, p = 0042, Mantel–Cox test). N = 5 mice/treatment group for all arms of the experiments. Raw data behind graphs are included in F of S1 Data. BM, bone marrow; PB, peripheral blood; Tam, tamoxifen; Veh, vehicle.
Fig 10.
Summary model of effects of Zeb1, Zeb2, and Zeb1/2 double deletion on hematopoietic system development and steady-state hematopoiesis.
(A) For comparative purposes, a more “classical” view of normal hematopoietic hierarchy is presented with more modern multipotent progenitor nomenclature (MPP1-4) highlighted in red. (B) Overview of Zeb2 null adult hematopoietic phenotypes previously described [9]. Adult mice develop myeloproliferative disease over time that is driven by enhanced G-CSF responsiveness [9] as well as mild differentiation defects in multiple HSPC populations including increased LT-HSCs, increased MEPs, decreased GMPs as well as defects in mature hematopoietic populations including decreased RBC, megakaryocytes, monocytes, and B cells but expanded terminal granulocyte differentiation (red arrows). (C) Represents a hybrid summary view between results of this study (red arrows) along with those found by [63] (blue arrows). Unlike Zeb2 KOs LSK, ST-HSC and MPP numbers are down in Zeb1 hematopoietic null mice and display multilineage differentiation defects with decreased numbers of progenitors and mature hematopoietic cells particularly T cells with mice developing thymic atrophy [62]. HSPC phenotype is characterized by increased EpCAM expression with altered survival and metabolism profiles. (D) Inducible loss of both Zeb1 and Zeb2 leads to acute BM failure with mice succumbing to lethal cytopenia within 2 weeks. Block in LT-HSC differentiation observed in Zeb2 KO is exacerbated in Zeb1/2 DKO settings and multilineage blocks especially in erythroid and megakaryocyte lineages are more severe (increased size of red arrows). Molecular analysis of LSK progenitors highlighting altered migratory and metabolic pathways as well as improper activation of multiple lineage-specific programs normally only observed in mature myeloid and lymphoid cell types. Specific relevant genes that are up- or down-regulated are indicated and further elaborated on in the discussion. BM, bone marrow; DKO, double knockout; G-CSF, granulocyte colony-stimulating factor; HSPC, hematopoietic stem and progenitor cell; KO, knockout; LSK, Lin−Sca1+cKit+; LT-HSC, long-term HSC; NK, natural killer; RBC, red blood cell; ST-HSC, short-term HSC; wt, wild-type.