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.

The first hematopoietic stem cells (HSCs) are formed through endothelial-to-hematopoietic transition (EHT) events during embryonic development. The transcription factor GATA2 is a crucial regulator of EHT and HSC function throughout life. Because GATA2 haploinsufficiency patients have inborn mutations, prenatal defects are likely to have an influence on disease development. In mice, Gata2 haploinsufficiency (Gata2+/-) reduces the number and the functionality of embryonic hematopoietic stem and progenitor cells (HSPCs) generated through EHT. However, the embryonic HSPC pool is heterogeneous and the mechanisms underlying this defect in Gata2+/- embryos are unclear. Here, we investigated whether Gata2 haploinsufficiency selectively affects a cellular subset undergoing EHT. We show that Gata2+/- HSPCs initiate but cannot fully activate hematopoietic programming during EHT. In addition, due to reduced activity of the endothelial repressor Gfi1b, Gata2+/- HSPCs cannot repress the endothelial identity to complete maturation. Finally, we show that hematopoietic-specific induction of gfi1b can restore HSC production in gata2b-null (gata2b-/-) zebrafish embryos. This study illustrates pivotal roles of Gata2 on the regulation of transcriptional network governing HSPC identity throughout EHT.

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.

Background: Paroxysmal nocturnal haemoglobinuria (PNH) is a life-threatening clinical syndrome caused by acquired mutations in PIGA in the haemopoietic compartment. The clinicopathological features of PNH can overlap with aplastic anaemia (AA) and myelodysplastic syndrome (MDS) and these entities may represent different manifestations of a common underlying pathological process. To date, the landscape of acquired mutations in de novo PNH (i.e. not preceded by a formal diagnosis of AA/MDS) remains incompletely characterised. Aims: To characterise the spectrum of acquired mutations in the cellular and cell-free compartments in a cohort of patients with de novo PNH. Methods: DNA was extracted from peripheral blood leukocytes (n = 19) or bone marrow (n = 2) from 21 patients with a clinicopathological diagnosis of PNH (defined as a detectable PNH clone by flow cytometry, active haemolysis and not meeting formal diagnostic criteria for MDS or AA). Patients with a pre-existing diagnosis of AA or MDS were excluded. Cell-free DNA (cfDNA) was also collected from 19 patients. DNA sequencing libraries were prepared from cellular DNA using both the Peter MacCallum Cancer Centre PanHaem hybridisation and Myeloid amplicon panels (sensitivity 5%) which includes sequence variant detection for recurrently mutated genes associated with haematological malignancy and whole genome copy number assessment. A custom panel using Anchored Multiplex PCR target enrichment chemistry and unique molecular barcodes was used for cfDNA sequencing (sensitivity <1%). Results: At time of sampling 18 of 21 patients were two or more years post diagnosis (median time since diagnosis of 8 years) with a median age of 41 years (range 23 -82). 16 patients were being treated with complement inhibitor therapy. PIGA mutations were detected in 19 patients, with multiple mutations detected in half the cohort (11 patients, range 2 -9 PIGA mutations per patient). 21 mutations were detected in the cellular DNA, while cfDNA sequencing revealed 30 additional PIGA mutations at low allelic burden (median allele frequency 2.19%, range 0.57% -4.86%) (20 frameshift, 15 missense, 10 splice site, 6 nonsense). Two patients did not have a detectable PIGA mutation but instead harboured focal copy number losses at the PIGA locus, which also involved ZRSR2 in the minimal deleted region. Remarkably, 7 of 21 patients had truncating ASXL1 mutations detectable in both the cellular and cfDNA (9 frameshift, 3 nonsense); in 4 patients multiple ASXL1 mutations were detected. Clonal pathogenic mutations were also detected in U2AF1 (2 patients) and BCOR (1 patient). Patients with ASXL1 and/or U2AF1 mutations had significantly lower neutrophil counts (mean 1.7 vs 1.08, p = 0.021) compared to those without. Haemoglobin and platelet counts were not statistically different between the two groups. Summary/Conclusion: Multiple subclonal PIGA mutations are detectable in patients with PNH. In addition acquired pathogenic variants in ASXL1, U2AF1 and ZRSR2 were detected in a third of the cohort. L...