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 CD45^lowc-Kit^high 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.

在妊娠中期小鼠胚胎期间，含有造血干细胞的主动脉内造血细胞簇 (IAHC) 出现在主动脉-性腺-中肾 (AGM) 区域的背主动脉中。我们之前表明，作为 IAHC 组成部分的CD45^低c-Kit^高细胞中的 Sox17 转导维持了簇形成和未分化状态，但 Sox17 形成簇的机制尚未阐明。通过微阵列基因表达分析，我们发现血管内皮钙粘蛋白 (VE-cad) 和内皮细胞选择性粘附分子 (ESAM) 的基因在 Sox17 转导的 c-Kit ^{+ 中}高水平表达细胞。在这里，我们通过体外实验展示了这些粘附分子在 IAHC 形成和维持未分化状态中的功能作用。我们通过整体免疫组织化学检测了 E10.5 胚胎背主动脉内皮细胞和 IAHC 中 VE-cad 和 ESAM 的表达。VE-cad中等表达水平和ESAM低表达水平的细胞具有最高的集落形成能力。Sox17-ERT 融合蛋白的他莫昔芬依赖性核易位诱导细胞簇的形成和Cdh5 (VE-cad)和ESAM基因的表达。我们展示了Cdh5 (VE-cad)和ESAM的诱导分别通过荧光素酶测定和染色质免疫沉淀测定来分析 Sox17 的表达和与其启动子的直接相互作用。此外，在 Sox17 转导的细胞中，shRNA 介导的Cdh5 (VE-cad)或ESAM基因敲低降低了多系集落形成潜力。这些发现表明 VE-cad 和 ESAM 在 IAHC 的高造血活性和簇形成中起重要作用。