The differentiation of hematopoietic stem cells (HSCs) is tightly controlled to ensure a proper balance between myeloid and lymphoid cell output. GATA2 is a pivotal hematopoietic transcription factor required for generation and maintenance of HSCs. GATA2 is expressed throughout development, but because of early embryonic lethality in mice, its role during adult hematopoiesis is incompletely understood. Zebrafish contains 2 orthologs of GATA2: Gata2a and Gata2b, which are expressed in different cell types. We show that the mammalian functions of GATA2 are split between these orthologs. Gata2b-deficient zebrafish have a reduction in embryonic definitive hematopoietic stem and progenitor cell (HSPC) numbers, but are viable. This allows us to uniquely study the role of GATA2 in adult hematopoiesis. gata2b mutants have impaired myeloid lineage differentiation. Interestingly, this defect arises not in granulocyte-monocyte progenitors, but in HSPCs. Gata2b-deficient HSPCs showed impaired progression of the myeloid transcriptional program, concomitant with increased coexpression of lymphoid genes. This resulted in a decrease in myeloid-programmed progenitors and a relative increase in lymphoid-programmed progenitors. This shift in the lineage output could function as an escape mechanism to avoid a block in lineage differentiation. Our study helps to deconstruct the functions of GATA2 during hematopoiesis and shows that lineage differentiation flows toward a lymphoid lineage in the absence of Gata2b.

Mutations in ELANE cause severe congenital neutropenia (SCN), but how they affect neutrophil production and contribute to leukemia predisposition is unknown. Neutropenia is alleviated by CSF3 (granulocyte colony-stimulating factor) therapy in most cases, but dose requirements vary between patients. Here, we show that CD34+CD45+ hematopoietic progenitor cells (HPCs) derived from induced pluripotent stem cell lines from patients with SCN that have mutations in ELANE (n = 2) or HAX1 (n = 1) display elevated levels of reactive oxygen species (ROS) relative to normal iPSC-derived HPCs. In patients with ELANE mutations causing misfolding of the neutrophil elastase (NE) protein, HPCs contained elevated numbers of promyelocyte leukemia protein nuclear bodies, a hallmark of acute oxidative stress. This was confirmed in primary bone marrow cells from 3 additional patients with ELANE-mutant SCN. Apart from responding to elevated ROS levels, PML controlled the metabolic state of these ELANE-mutant HPCs as well as the expression of ELANE, suggestive of a feed-forward mechanism of disease development. Both PML deletion and correction of the ELANE mutation restored CSF3 responses of these ELANE-mutant HPCs. These findings suggest that PML plays a crucial role in the disease course of ELANE-SCN characterized by NE misfolding, with potential implications for CSF3 therapy.

The transcription factor GATA2 has pivotal roles in hematopoiesis. Germline GATA2 mutations result in GATA2 haploinsufficiency characterized by immunodeficiency, bone marrow failure and predispositions to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Clinical symptoms in GATA2 patients are diverse and mechanisms driving GATA2 related phenotypes are largely unknown. To explore the impact of GATA2 haploinsufficiency on hematopoiesis, we generated a zebrafish model carrying a heterozygous mutation in gata2b, an orthologue of GATA2. Morphological analysis revealed progression of myeloid and erythroid dysplasia in gata2b+/- kidney marrow (KM). Single cell RNA sequencing on KM cells showed that the erythroid dysplasia in gata2b+/- zebrafish was preceded by a differentiation block in erythroid progenitors, hallmarked by downregulation of cytoskeletal transcripts, aberrant proliferative signatures and ribosome biogenesis. Additionally, transcriptional and functional analysis of Gata2b haploinsufficient hematopoietic stem cells (HSCs) indicated that proliferative stress within the HSC compartment possibly contributes to the development of myeloid and erythroid dysplasia in gata2b+/- zebrafish.

Hematopoietic stem cells (HSCs) are tightly controlled to maintain a balance between myeloid and lymphoid cell differentiation. Gata2 is a pivotal hematopoietic transcription factor required for HSC generation and maintenance. We generated a zebrafish mutant for the mouse Gata2 orthologue, gata2b. We found that in adult zebrafish, gata2b is required for both neutrophilic-and monocytic lineage differentiation. Single cell transcriptome analysis revealed that the myeloid defect present in Gata2b deficient zebrafish arise in the most immature hematopoietic stem and progenitor cell (HSPC) compartment and that this population is instead committed towards the lymphoid and erythroid lineage. Taken together, we find that Gata2b is vital for the fate choice between the myeloid and lymphoid lineages.