Gata2 is a key transcription factor required to generate Haematopoietic Stem and Progenitor Cells (HSPCs) from haemogenic endothelium (HE); misexpression of Gata2 leads to haematopoietic disorders. Here we deleted a conserved enhancer (i4 enhancer) driving pan-endothelial expression of the zebrafish gata2a and showed that Gata2a is required for HE programming by regulating expression of runx1 and of the second Gata2 orthologue, gata2b. By 5 days, homozygous gata2a Δi4/Δi4 larvae showed normal numbers of HSPCs, a recovery mediated by Notch signalling driving gata2b and runx1 expression in HE. However, gata2a Δi4/Δi4 adults showed oedema, susceptibility to infections and marrow hypo-cellularity, consistent with bone marrow failure found in GATA2 deficiency syndromes. Thus, gata2a expression driven by the i4 enhancer is required for correct HE programming in embryos and maintenance of steady-state haematopoietic stem cell output in the adult. These enhancer mutants will be useful in exploring further the pathophysiology of GATA2-related deficiencies in vivo.

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 enteric nervous system (ENS) regulates many gastrointestinal functions including peristalsis, immune regulation and uptake of nutrients. Defects in the ENS can lead to severe enteric neuropathies such as Hirschsprung disease (HSCR), which is caused by defective ENS development. Zebrafish have proven to be fruitful in the identification of novel genes involved in ENS development and HSCR pathology. However, the composition and specification of enteric neurons and glial subtypes of the larval zebrafish at a single cell level, remains mainly unexplored. Here, we performed single cell RNA sequencing of zebrafish ENS at 5 days post-fertilization. We identified both vagal neural crest progenitors and Schwann cell precursors, as well as four clusters of early differentiated neurons. Interestingly, since we took an unbiased approach where we sequenced total intestines, an elavl3+/phox2bb- population of neurons and the presence of cx43+/phox2bb- enteric glia were identified in larval zebrafish. These populations have not been described before. Pseudotime analysis supported binary neurogenic branching of ENS differentiation, which happens via a notch-responsive state. Together, our data revealed previously unrecognized ENS populations and serve as a resource to gain new insights on ENS development and specification, proving that the zebrafish is a valuable model organism in the quest towards understanding and treating congenital enteric neuropathies.

Haematopoietic stem and progenitor cells (HSPCs) maintain the vertebrate blood system throughout life and their emergence from haemogenic endothelium (HE) is tightly regulated by transcription factors such as Gata2. Zebrafish have two orthologues of Gata2, gata2a and gata2b, the latter required for HSPC emergence. Here we deleted a conserved enhancer driving gata2a expression in endothelium (i4 enhancer) and showed that Gata2a is required for HE programming by regulating expression of gata2b and runx1. By 5 days, homozygous gata2a Δi4/Δi4 larvae showed normal numbers of HSPCs, a recovery mediated by Notch signalling driving gata2b expression in HE. However, gata2a Δi4/Δi4 adults showed lymphoedema, susceptibility to infections and marrow hypocellularity, consistent with bone marrow failure of MonoMAC syndrome patients. Thus, Gata2a is required for HE programming and haematopoiesis in the adult. Like MonoMAC syndrome patients, gata2a Δi4/Δi4 mutants developed acute myeloid leukemia. These mutants will be invaluable to explore the pathophysiology of MonoMAC syndrome in vivo.

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.

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