Flow Cytometry data from: "The EMT transcription factor Zeb1 is essential for HSPC differentiation that acts synergistically with Zeb2 in fine-tuning hematopoietic lineage fidelity"

Abstract:

The Zeb2 transcription factor has been demonstrated to play important roles in hematopoiesis and leukemic transformation. Zeb1 is a close family member of Zeb2 but has remained more enigmatic concerning its roles in hematopoiesis. Here we show using conditional loss of function approaches and bone marrow reconstitution experiments that Zeb1 plays cell autonomous role in hematopoietic lineage differentiation, particularly as a positive regulator of monocyte development in addition to its previously reported important role in T-cell differentiation. Analysis of existing single cell RNAseq data of early hematopoiesis has revealed distinctive expression differences between Zeb1 and Zeb2 in HSPC differentiation with Zeb2 being more highly and broadly expressed that Zeb1 except at a key transition point (ST-HSCàMPP1) whereby Zeb1 appears to be the dominantly expressed family member. Inducible deletion of both Zeb1 and Zeb2 using a tamoxifen inducible Cre-mediated approach leads to acute bone marrow failure at this transition point with increased long-term and shortterm hematopoietic stem cell numbers and an accompanying decrease in all hematopoietic lineage differentiation. Bioinformatics analysis of RNAseq data has revealed that Zeb2 acts predominantly as a transcriptional repressor involved in restraining mature hematopoietic lineage gene expression programs from being expressed too early in hematopoietic stem and progenitor cells (HSPCs). Zeb1 appears to fine tune this repressive role during hematopoiesis to ensure hematopoietic lineage fidelity. Analysis of ROSA26 locus based transgenic models has revealed that Zeb1 as well as Zeb2 overexpression within the hematopoietic system can drive extramedullary hematopoiesis/splenomegaly and enhanced monocyte development. Finally, deletion of Zeb2 alone or Zeb1/2 together was found to enhance survival in secondary MLL-AF9 AML models attesting to the oncogenic role of Zeb1/2 in AML.

Flow cytometric and Hematocrit analysis methods: 

 Cells were stained with antibodies listed in the provided Supplemental Table (Antibodies.xlsx) according to the  manufacturer guidelines. Flow cytometric analyses were performed on the LSRII and Fortessa  X-20 cytometer (BD Biosciences) and the results were analysed by FACSDiva or FlowJo software (BD Biosciences). Cells for MLL-AF9 experiments and RNA-seq were stained and  sorted on Influx or FACSAria Fusion sorters (BD Biosciences) at AMREP Flow Cytometry  Core Facility and FlowCore, Monash University. 
Submandibular blood samples were collected into EDTA-coated tubes, and hematology parameters were measured using a HemaVet 950FS automated blood analysis machine (Drew Scientific).