Artery Specific Enhancer Is Essential for Specification of Arterial Hemogenic Endothelium with T Lymphoid Potential

Hematopoietic development in the vertebrate embryo occurs in multiple waves from hemogenic endothelium (HE) intermediates in several extra- and intra-embryonic locations including yolk sac vessels, the aorta, vitelline and umbilical arteries, placenta, endocardium, and possibly head and somitic vasculature. Although the earliest waves of hematopoiesis, primitive and erythromyeloid, are NOTCH-independent and occur in different vessel types, initiation of HSC and lymphoid cell formation requires NOTCH signaling and only occurs in arteries. During vascular development, arterial fate is specified following induction of DLL4 expression by VEGF. DLL4 expression is initiated by signaling through a highly conserved VEGF-responsive artery-specific enhancer located within the third intron of DLL4 (DLL4i3) which is controlled by ETS, SOXF and RBPJ factors. To define the role of DLL4i3 enhancer in hematopoietic development, we generated DLL4i3-Venus reporter and knockout (DLL4^Di3/Di3) human pluripotent stem cells (hPSCs) using CRISPR-Cas9 mediated technology. Using a 3-dimensional, organoid-like differentiation system (Spheroid) we demonstrated that DLL4i3 enhancer activity recapitulates endogenous DLL4 expression and arterial endothelial cell specification in human iPSC cultures as studied by single cell RNAseq analysis and flow cytometry. Through time kinetic analysis of enhancer activity, we show that the DLL4 expression starts appearing in HE and is highest at the DLL4⁺CXCR4⁺ HE stage. Moreover, DLL4i3-Venus⁺ HE and hematopoietic progenitors produced more myeloid CFCs and T cells as compared DLL4i3-Venus^- hemogenic cells. Deletion of DLL4i3 enhancer impaired arterial specification from hPSCs and showed diminished formation of DLL4⁺CXCR4^+/- HE. In contrast to wild type (WT), HE generated from DLL4^Di3/Di3 hPSCs showed impaired CFC potential lacking G-CFCs and markedly diminished T cell potential when cocultured on OP9-DLL4. However, deletion of the DLL4i3 enhancer had no significant effect on NK cell differentiation. Coculture of DLL4^Di3/Di3 HE on OP9-DLL4 restored myeloid CFC potential to the levels of WT HE. In differentiation cultures composed of equal mix of DLL4^i3+/Di3+-GFP and DLL4^Di3/Di3 hPSCs, T cell potential of DLL4^Di3/Di3 HE was only partially restored. Analysis of NOTCH signaling following culture of HE on plates coated with DLL4-Fc revealed impaired NOTCH activation in DLL4^Di3/Di3 HE. Single cell RNAseq analysis comparing DLL4i3-Venus reporter and DLL4^Di3/Di3 HE and hematopoietic progenitors identified substantial differences in the dynamic transcriptional signature of blood cells during arterialization of the HE. Overall, we established that DLL4 is required for arterial programming of HE with lymphomyeloid potential, and this function requires the activity of DLL4i3 enhancer.

Slukvin:Cynata Therapeutics: Consultancy, Current holder of stock options in a privately-held company; Umoja: Consultancy.