GATA2 Is Essential for Endothelial-to-Hematopoietic Transition but Does Not Affect Formation of Hemogenic Endothelium

The formation of blood cells from hemogenic endothelium (HE) is a key element of leading to establishment of the hematopoietic system during embryogenesis. It has become increasingly clear that HE represents a distinct lineage of vascular endothelium with a capacity to undergo endothelial-to-hematopoietic transition (EHT). However, the processes guiding HE specification and EHT are poorly understood. The GATA2 transcription factor is of particular interest since it is critical for development of the entire hematopoietic system including hematopoietic stem cells (HSCs) during embryogenesis. Although GATA2 knockout has been shown to abrogate formation of intraortic hematopoietic clusters, it is not clear whether GATA2 is required for HE formation, EHT per se, or if it controls HE specification and EHT in a non-cell autonomous manner. To study the function of GATA2 during hematopoietic development, we engineered a GATA2 knockout H1 human embryonic stem cell (hESC) carrying doxycycline (DOX)-inducible GATA2 transgene. We demonstrated that GATA2 deficiency dramatically impaired definitive and primitive hematopoiesis from hESCs. To define the stage of development affected by GATA2, we treated iG2^-/- and iG2^+/+ hESCs with DOX in stepwise fashion, and evaluated the stage of development at which GATA2 induction has the most profound effect on hematopoiesis. We found that DOX treatment has the most dramatic effect on production of CD43⁺ cells and CFC potential when performed on days 3-4 or 4-5 of differentiation. In contrast, DOX treatment on days 0-2 suppressed differentiation, while treatment on days 5-6 has little to no effect. Since formation of HE and EHT in our system occurs during days 4-5 of differentiation, i.e. when we see the most dramatic effect of DOX treatment, we concluded that GATA2 may be important for HE formation or EHT. Flow cytometric analysis revealed that formation of VE-cadherin+CD43-CD73- HE, including DLL4⁺ arterial HE subset, and non-HE was not affected in iG2^-/- hESCs, or following GATA2 induction with DOX. Next, we isolated iG2^-/- HE from day 4 of differentiation and assessed how DOX treatment affected blood formation. In the absence of DOX, iG2^-/-HE produced very few blood cells, while DOX treatment restored blood formation from HE. No significant difference was found in proliferation and in apoptosis of hematopoietic progenitors following EHT, regardless of the presence of GATA2, implying the specific effect of GATA2 on EHT.

To define molecular pathways which involved in EHT impairment in iG2^-/- cells, we performed RNAseq analysis of HE in DOX- and DOX+ conditions. Gene set enrichment analysis (GSEA) detected significant upregulation of sets of genes responding to TGFβ stimulation in iG2^-/- HE on day 1 of secondary culture without DOX as compared to DOX treated cells, thereby suggesting that upregulation of target genes of TGFβ signaling may contribute to EHT impairment in GATA2-deficient cells. Indeed, treatment of iG2^-/- HE with TGFβ-inhibitor significantly increased EHT and CD43⁺ hematopoietic progenitor production with CFU-G and GM potential. Overall, our studies suggest that GATA2 is dispensable for specification of HE, but is essential for EHT and proper hematopoietic specification. In addition, inhibiting TGFβ signaling partially restored the GATA2-dependent EHT in the absence of GATA2.