Migueles Rosa Portero, Shaw Louise, Rodrigues Neil P, May Gillian, Henseleit Korinna, Anderson Kathryn G V, Goker Hakan, Jones C Michael, de Bruijn Marella F T R, Brickman Joshua M, Enver Tariq
MRC Centre for Regenerative Medicine - Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, 5 Little France Drive, EH16 4UU Edinburgh, UK.
MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK.
Dev Biol. 2017 Apr 15;424(2):236-245. doi: 10.1016/j.ydbio.2016.12.021. Epub 2017 Feb 9.
Hematopoietic stem cells (HSCs) emerge during development via an endothelial-to-hematopoietic transition from hemogenic endothelium of the dorsal aorta (DA). Using in situ hybridization and analysis of a knock-in RedStar reporter, we show that the transcriptional regulator Hhex is expressed in endothelium of the dorsal aorta (DA) and in clusters of putative HSCs as they are specified during murine development. We exploited this observation, using the Hhex locus to define cis regulatory elements, enhancers and interacting transcription factors that are both necessary and sufficient to support gene expression in the emerging HSC. We identify an evolutionarily conserved non-coding region (ECR) in the Hhex locus with the capacity to bind the hematopoietic-affiliated transcriptional regulators Gata2, SCL, Fli1, Pu.1 and Ets1/2. This region is sufficient to drive the expression of a transgenic GFP reporter in the DA endothelium and intra-aortic hematopoietic clusters. GFP-positive AGM cells co-expressed HSC-associated markers c-Kit, CD34, VE-Cadherin, and CD45, and were capable of multipotential differentiation and long term engraftment when transplanted into myelo-ablated recipients. The Hhex ECR was also sufficient to drive expression at additional blood sites including the yolk sac blood islands, fetal liver, vitelline and umbilical arteries and the adult bone marrow, suggesting a common mechanism for Hhex regulation throughout ontogenesis of the blood system. To explore the physiological requirement for the Hhex ECR region during hematoendothelial development, we deleted the ECR element from the endogenous locus in the context of a targeted Hhex-RedStar reporter allele. Results indicate a specific requirement for the ECR in blood-associated Hhex expression during development and further demonstrate a requirement for this region in the adult HSC compartment. Taken together, our results identified the ECR region as an enhancer both necessary and sufficient for gene expression in HSC development and homeostasis. The Hhex ECR thus appears to be a core node for the convergence of the transcription factor network that governs the emergence of HSCs.
造血干细胞（HSCs）在发育过程中通过背主动脉（DA）的造血内皮细胞发生内皮向造血的转变而产生。通过原位杂交和对敲入红星报告基因的分析，我们发现转录调节因子Hhex在背主动脉（DA）的内皮细胞以及小鼠发育过程中特定阶段的假定造血干细胞簇中表达。我们利用这一观察结果，通过Hhex基因座来定义顺式调控元件、增强子以及相互作用的转录因子，这些对于支持新兴造血干细胞中的基因表达是必要且充分的。我们在Hhex基因座中鉴定出一个进化上保守的非编码区域（ECR），它能够结合造血相关转录调节因子Gata2、SCL、Fli1、Pu.1和Ets1/2。该区域足以驱动转基因GFP报告基因在DA内皮细胞和主动脉内造血簇中的表达。GFP阳性的主动脉-性腺-中肾（AGM）区细胞共表达造血干细胞相关标志物c-Kit、CD34、血管内皮钙黏蛋白（VE-Cadherin）和CD45，并且在移植到骨髓消融受体后能够进行多能分化和长期植入。Hhex ECR也足以在包括卵黄囊血岛、胎肝、卵黄和脐动脉以及成年骨髓在内的其他血液部位驱动表达，这表明在整个血液系统的个体发育过程中，Hhex调控存在共同机制。为了探究Hhex ECR区域在血液内皮细胞发育过程中的生理需求，我们在靶向Hhex-红星报告基因等位基因的背景下从内源性基因座中删除了ECR元件。结果表明在发育过程中血液相关的Hhex表达对ECR有特定需求，并且进一步证明了该区域在成年造血干细胞区室中的需求。综上所述，我们的结果确定ECR区域是造血干细胞发育和稳态中基因表达所必需且充分的增强子。因此，Hhex ECR似乎是控制造血干细胞出现的转录因子网络汇聚的核心节点。
