A Sox17 downstream geneRasip1is involved in the hematopoietic activity of intra-aortic hematopoietic clusters in the midgestation mouse embryo

AbstractBackgroundDuring mouse embryonic development, definitive hematopoiesis is first detected around embryonic day (E) 10.5 in the aorta-gonad-mesonephros (AGM) region. Hematopoietic stem cells (HSCs) arise in the dorsal aorta’s intra-aortic hematopoietic cell clusters (IAHCs). We have previously reported that a transcription factor Sox17, is expressed in IAHCs, and that, among them, CD45lowc-Kithighcells have high hematopoietic activity. Furthermore, forced expression of Sox17 in this population of cells can maintain the formation of hematopoietic cell clusters. However, how Sox17 does so, particularly downstream signaling involved, remains poorly understood. The purpose of this study is to search for new Sox17 targets which contribute to cluster formation with hematopoietic activity.MethodsRNA-sequencing (RNA-seq) analysis was done to identify genes that are up- regulated in Sox17-expressing IAHCs as compared with Sox17-negative ones. Among the top 7 highly expressed genes, Rasip1 which had been reported to be a vascular-specific regulator was focused on in this study and firstly the whole-mount immunostaining was done. We conducted a luciferase reporter assay to identify the Sox17 binding site for Rasip1 gene induction. We also analyzed the cluster formation and the multi-lineage colony forming ability of Rasip1-transduced cells and Rasip1-knockdown Sox17-transduced cells.ResultsThe increase of theRasip1expression level was observed in Sox17-positive CD45lowc-Kithighcells as compared with the Sox17-nonexpressing control. Also, the expression level of theRasip1gene was increased by the Sox17-nuclear translocation. Rasip1 was expressed on the membrane of IAHCs, overlapping with the endothelial cell marker, CD31, and hematopoietic stem/progenitor marker (HSPC), c-Kit. Overexpression of Rasip1 in CD45lowc-Kithighcells led to a significant but transient increase in hematopoietic activity, while Rasip1 knock-down in Sox17-transduced cells decreased the cluster formation and diminished the colony-forming ability.ConclusionsRasip1 knockdown in Sox17-transduced CD45lowc-Kithighcells displayed a significant decrease in the multi-lineage colony forming ability and the cluster size. Rasip1 overexpression in Sox17-untransduced CD45lowc-Kithighcells led to a significant but transient increase in the multi-lineage colony forming ability, suggesting the presence of a cooperating factor for sustained hematopoietic activity.