3151 – SINGLE-CELL RNA SEQUENCING ANALYSIS REVEALS ROLE FOR GATA3 IN THE AORTIC ENDOTHELIUM AT THE TIME OF HEMATOPOIETIC STEM CELL EMERGENCE

Abstract

The endothelial-to-haematopoietic transition (EHT) gives rise to the first murine haematopoietic stem cells (HSCs) in the aorta-gonads-mesonephros (AGM) region around E10.5. While our understanding of the transcriptional networks regulating HSC emergence is ever-increasing, the mechanisms governing the EHT process are still being investigated. The transcription factor Gata3, whose function has previously been characterized in the sympathetic nervous system AGM niche, is expressed directly in the haemogenic endothelium prior to EHT. Previous studies have shown that Gata3-expressing endothelial cells (ECs) have a greater capacity to produce haematopoietic colonies, over Gata3-negative ECs, while endothelial-specific Gata3 knockout leads to a greatly decreased ability to repopulate AGM-transplant recipient mice. Single-cell RNA sequencing analysis of Gata3 knockout ECs sorted from murine AGMs reveals an upregulation of factors involved in endothelial cell fate differentiation, such as Pecam1, and a dysregulation of adhesion molecule genes, including increased expression of Epcam and Vcam1, all of which are likely to disrupt the normal EHT process. Furthermore, downregulation of critical stem cell proliferation regulator Kitl may explain the negative effect of Gata3 knockout on transplanted AGMs. Another key finding has been the identification of cell surface antigens CD200 and CD200r1 as new markers for the enrichment of haemogenic cells via trajectory analysis of Gata3+ endothelial cells differentiating into pre-HSCs. CD200, which is expressed on arterial endothelium, contains the CD41lo cells capable of engrafting after transplantation, while CD200r1 is expressed within the pre-HSC compartment, but only CD200r1-negative cells are able to repopulate. These markers will undoubtedly prove useful in future studies attempting to isolate HSC precursors from murine AGMs. The endothelial-to-haematopoietic transition (EHT) gives rise to the first murine haematopoietic stem cells (HSCs) in the aorta-gonads-mesonephros (AGM) region around E10.5. While our understanding of the transcriptional networks regulating HSC emergence is ever-increasing, the mechanisms governing the EHT process are still being investigated. The transcription factor Gata3, whose function has previously been characterized in the sympathetic nervous system AGM niche, is expressed directly in the haemogenic endothelium prior to EHT. Previous studies have shown that Gata3-expressing endothelial cells (ECs) have a greater capacity to produce haematopoietic colonies, over Gata3-negative ECs, while endothelial-specific Gata3 knockout leads to a greatly decreased ability to repopulate AGM-transplant recipient mice. Single-cell RNA sequencing analysis of Gata3 knockout ECs sorted from murine AGMs reveals an upregulation of factors involved in endothelial cell fate differentiation, such as Pecam1, and a dysregulation of adhesion molecule genes, including increased expression of Epcam and Vcam1, all of which are likely to disrupt the normal EHT process. Furthermore, downregulation of critical stem cell proliferation regulator Kitl may explain the negative effect of Gata3 knockout on transplanted AGMs. Another key finding has been the identification of cell surface antigens CD200 and CD200r1 as new markers for the enrichment of haemogenic cells via trajectory analysis of Gata3+ endothelial cells differentiating into pre-HSCs. CD200, which is expressed on arterial endothelium, contains the CD41lo cells capable of engrafting after transplantation, while CD200r1 is expressed within the pre-HSC compartment, but only CD200r1-negative cells are able to repopulate. These markers will undoubtedly prove useful in future studies attempting to isolate HSC precursors from murine AGMs.