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GATA2 Promotes Hematopoietic Development and Represses Cardiac Differentiation of Human Mesoderm.

ABSTRACT: In vertebrates, GATA2 is a master regulator of hematopoiesis and is expressed throughout embryo development and in adult life. Although the essential role of GATA2 in mouse hematopoiesis is well established, its involvement during early human hematopoietic development is not clear. By combining time-controlled overexpression of GATA2 with genetic knockout experiments, we found that GATA2, at the mesoderm specification stage, promotes the generation of hemogenic endothelial progenitors and their further differentiation to hematopoietic progenitor cells, and negatively regulates cardiac differentiation. Surprisingly, genome-wide transcriptional and chromatin immunoprecipitation analysis showed that GATA2 bound to regulatory regions, and repressed the expression of cardiac development-related genes. Moreover, genes important for hematopoietic differentiation were upregulated by GATA2 in a mostly indirect manner. Collectively, our data reveal a hitherto unrecognized role of GATA2 as a repressor of cardiac fates, and highlight the importance of coordinating the specification and repression of alternative cell fates.

SUBMITTER: Castano J

PROVIDER: S-EPMC6742600 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The development of the hematopoietic system involves multiple cellular steps beginning with the formation of the mesoderm from the primitive streak, followed by emergence of precursor populations that become committed to either the endothelial or hematopoietic lineages. A number of growth factors such as activins and fibroblast growth factors (FGFs) are known to regulate the early specification of hematopoietic fated mesoderm, notably in amphibians. However, the potential roles of these factors in the development of mesoderm and subsequent hematopoiesis in the human have yet to be delineated. Defining the cellular and molecular mechanisms by which combinations of mesoderm-inducing factors regulate this stepwise process in human cells in vitro is central to effectively directing human embryonic stem cell (hESC) hematopoietic differentiation. Herein, using hESC-derived embryoid bodies (EBs), we show that Activin A, but not basic FGF/FGF2 (bFGF), promotes hematopoietic fated mesodermal specification from pluripotent human cells. The effect of Activin A treatment relies on the presence of bone morphogenetic protein 4 (BMP4) and both of the hematopoietic cytokines stem cell factor and fms-like tyrosine kinase receptor-3 ligand, and is the consequence of 2 separate mechanisms occurring at 2 different stages of human EB development from mesoderm to blood. While Activin A promotes the induction of mesoderm, as indicated by the upregulation of Brachyury expression, which represents the mesodermal precursor required for hematopoietic development, it also contributes to the expansion of cells already committed to a hematopoietic fate. As hematopoietic development requires the transition through a Brachyury+ intermediate, we demonstrate that hematopoiesis in hESCs is impaired by the downregulation of Brachyury, but is unaffected by its overexpression. These results demonstrate, for the first time, the functional significance of Brachyury in the developmental program of hematopoietic differentiation from hESCs and provide an in-depth understanding of the molecular cues that orchestrate stepwise development of hematopoiesis in a human system.

Project description:GATA2 deficiency is an inherited or sporadic genetic disorder characterized by distinct cellular deficiency, bone marrow failure, various infections, lymphedema, pulmonary alveolar proteinosis, and predisposition to myeloid malignancies resulting from heterozygous loss-of-function mutations in the GATA2 gene. How heterozygous GATA2 mutations affect human hematopoietic development or cause characteristic cellular deficiency and eventual hypoplastic myelodysplastic syndrome or leukemia is not fully understood. We used induced pluripotent stem cells (iPSCs) to study hematopoietic development in the setting of GATA2 deficiency. We performed hematopoietic differentiation using iPSC derived from patients with GATA2 deficiency and examined their ability to commit to mesoderm, hemogenic endothelial precursors (HEPs), hematopoietic stem progenitor cells, and natural killer (NK) cells. Patient-derived iPSC, either derived from fibroblasts/marrow stromal cells or peripheral blood mononuclear cells, did not show significant defects in committing to mesoderm, HEP, hematopoietic stem progenitor, or NK cells. However, HEP derived from GATA2-mutant iPSC showed impaired maturation toward hematopoietic lineages. Hematopoietic differentiation was nearly abolished from homozygous GATA2 knockout (KO) iPSC lines and markedly reduced in heterozygous KO lines compared with isogenic controls. On the other hand, correction of the mutated GATA2 allele in patient-specific iPSC did not alter hematopoietic development consistently in our model. GATA2 deficiency usually manifests within the first decade of life. Newborn and infant hematopoiesis appears to be grossly intact; therefore, our iPSC model indeed may resemble the disease phenotype, suggesting that other genetic, epigenetic, or environmental factors may contribute to bone marrow failure in these patients following birth. However, heterogeneity of PSC-based models and limitations of in vitro differentiation protocol may limit the possibility to detect subtle cellular phenotypes.

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GATA2 Promotes Hematopoietic Development and Represses Cardiac Differentiation of Human Mesoderm.

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