RESUMEN
BACKGROUND: During 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-Kithigh cells 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. METHODS: RNA-sequencing (RNA-seq) analysis was done to identify genes that are upregulated 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 luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay to examine whether Sox17 regulates Rasip1 gene expression via binding to its enhancer element. We also analyzed the cluster formation and the multilineage colony-forming ability of Rasip1-transduced cells and Rasip1-knockdown Sox17-transduced cells. RESULTS: The increase of the Rasip1 expression level was observed in Sox17-positive CD45lowc-Kithigh cells as compared with the Sox17-nonexpressing control. Also, the expression level of the Rasip1 gene 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. Rasip1 expression was observed in most part of c-Kit+Sox17+ cells in IAHCs. Luciferase reporter assay and ChIP assay indicated that one of the five putative Sox17-binding sites in the Rasip1 enhancer region was important for Rasip1 expression via Sox17 binding. Rasip1 knockdown in Sox17-transduced cells decreased the cluster formation and diminished the colony-forming ability, while overexpression of Rasip1 in CD45lowc-Kithigh cells led to a significant but transient increase in hematopoietic activity. CONCLUSIONS: Rasip1 knockdown in Sox17-transduced CD45lowc-Kithigh cells displayed a significant decrease in the multilineage colony-forming ability and the cluster size. Rasip1 overexpression in Sox17-untransduced CD45lowc-Kithigh cells led to a significant but transient increase in the multilineage colony-forming ability, suggesting the presence of a cooperating factor for sustained hematopoietic activity.
RESUMEN
Hematopoietic stem cell-containing intra-aortic hematopoietic cell clusters (IAHCs) emerge in the dorsal aorta of the aorta-gonad-mesonephros (AGM) region during midgestation mouse embryos. We previously showed that transduction of Sox17 in CD45lowc-Kithigh cells, which are one component of IAHCs, maintained the cluster formation and the undifferentiated state, but the mechanism of the cluster formation by Sox17 has not been clarified. By microarray gene expression analysis, we found that genes for vascular endothelial-cadherin (VE-cad) and endothelial cell-selective adhesion molecule (ESAM) were expressed at high levels in Sox17-transduced c-Kit+ cells. Here we show the functional role of these adhesion molecules in the formation of IAHCs and the maintenance of the undifferentiated state by in vitro experiments. We detected VE-cad and ESAM expression in endothelial cells of dorsal aorta and IAHCs in E10.5 embryos by whole mount immunohistochemistry. Cells with the middle expression level of VE-cad and the low expression level of ESAM had the highest colony-forming ability. Tamoxifen-dependent nuclear translocation of Sox17-ERT fusion protein induced the formation of cell clusters and the expression of Cdh5 (VE-cad) and ESAM genes. We showed the induction of the Cdh5 (VE-cad) and ESAM expression and the direct interaction of Sox17 with their promoter by luciferase assay and chromatin immunoprecipitation assay, respectively. Moreover, shRNA-mediated knockdown of either Cdh5 (VE-cad) or ESAM gene in Sox17-transduced cells decreased the multilineage-colony forming potential. These findings suggest that VE-cad and ESAM play an important role in the high hematopoietic activity of IAHCs and cluster formation.
