小鼠胚胎头部造血功能的系统性研究

The developmental origin of hematopoietic stem cells (HSC) has been controversial for many years. As widely accepted, such cells are believed to generate in the intra-embryonic aorta-gonad-mesonephros region through the endothelial-hematopoietic transition. Recently, we revealed the HSC potential and fate of cerebrovascular endothelial cells in mouse embryos by HSC transplantation and lineage tracing. However, there are still several critical issues to be addressed. In this study, we will use pre-circulation (E8.25) or non-heartbeat (Ncx1 gene knockout) embryos to validate whether the HSC potential in head is independent of the embryonic circulation. Furthermore, different populations of cerebrovascular endothelial cells will be purified by immunophenotyping and temporally-limited genetic labeling, followed by comparing their hemogenic capacity (with lung endothelial cells as negative control). Then, the transcriptional factors with potential regulating effects will be selected by comparative transcriptome analysis. Finally, the regulatory capacities of these molecules on endothelial-hematopoietic transition will be functinally defined by various strategies. Generally, this study will greatly improve our understanding of cerebrovasculature hematopoiesis and ultimately provide unique and important clues on regeneration of hematopoietic system.

造血干细胞的发育起源一直存在很大争议。主流观点认为：造血干细胞主要通过内皮细胞造血转化的方式在小鼠胚内的主动脉-性腺-中肾区（AGM区）产生。近期我们通过长期移植、谱系示踪等手段证实小鼠胚胎脑血管内皮细胞的造血干细胞潜能和命运，但仍有若干关键科学问题亟待解答。本研究拟应用循环建立前胚胎（8.25天）、无心跳胚胎（Ncx1基因敲除），结合长期移植等方法明确胚胎头部的造血干细胞潜能是否独立于循环产生。此外，通过免疫表型、时间限定的遗传学标记获取多种脑血管内皮细胞亚群，比较其体外和体内的生血潜能（以肺血管内皮细胞为阴性对照），应用表达谱分析初步筛选出具有潜在调节功能的转录因子，并通过功能验证最终发掘出调控内皮细胞造血转化的核心分子。本研究将进一步完善脑血管造血功能的新发现，为造血系统再生提供独特、重要的策略指导。

造血干细胞的发育起源一直存在很大争议。主流观点认为：造血干细胞主要通过内皮细胞造血转化的方式在小鼠胚内的主动脉-性腺-中肾区（AGM区）产生。近期我们通过长期移植、谱系示踪等手段证实小鼠胚胎脑血管内皮细胞的造血干细胞潜能和命运，发现了一系列成果。. 首先，我们针对HSC发育过程中的具有代表性的5类细胞（血管内皮细胞，I型和II型造血干细胞前体，胚胎12天和胚胎14天的造血干细胞）进行单细胞转录组高通量测序，首次揭示了造血干细胞前体在转录活性、动静脉基因表达、代谢状态、信号通路和转录因子网络等方面的核心特征。并且我们通过与更多细胞类群的单细胞转录组测序数据的深入对比分析，发掘出了造血干细胞前体特异表达的98个核心基因。该部分工作发表在2016年的Nature杂志上。. 在此基础上，我们进一步揭示了第E11天小鼠胚胎发育中最早产生的造血干细胞以及造血干细胞前体具有显著的功能异质性，从而完成整个生命周期造血干细胞功能异质性的图谱绘制。该工作发表于2017年的Cell Research上。