HEMATOPOIETIC COMMITMENT: MOLECULAR MECHANISMS

描述(由申请者提供)：我们的长期目标是了解在发育中的胚胎中造血系统是如何建立的。ER71、FlI1、Erg、Ets1、Ets2、Elk3和TELETS转录因子以及GATA2和scl与胚胎发育过程中的造血和血管发育相关。它们在造血细胞和内皮细胞中表达。此外，当这些基因中的任何一个被灭活时，小鼠都会表现出不同程度的造血和血管缺陷。显然，在ETS、GATA2和SCL下游的基因调控网络中存在重叠，我们需要了解这一点。事实上，新出现的研究认为ETS-GATA-Ebox基序对造血发育至关重要。例如，RUNX1在造血干细胞中的表达受ETS-GATA-Ebox基序的调控。至于ETS因子，目前还不清楚ETS因子的特异性是如何确定的，也不清楚在造血和血管发育过程中ETS因子之间存在多少功能冗余。我们有令人兴奋的初步数据表明，存在ETS等级，ER71处于顶端。值得注意的是，与其他ETS因子相比，ER71缺乏会导致最严重的造血和血管缺陷，GATA2或SCL缺乏。有趣的是，ER71在胚胎发育过程中的表达是短暂的：它在背主动脉的原始条纹和大血管中表达，在那里特定的造血程序被激活。后期卵黄囊、胎肝或 骨髓中，ER71的表达大大降低，但其他ETS因子的表达显著。根据这些数据，我们设想ER71诱导ETS-GATA2-SCL三联体介导的造血程序激活。在这个方案中，我们检验了以下假设：1)ER71在Ets转录因子中处于造血程序诱导的顶端；2)Ets转录 3)ETS-GATA2-SCL三联体是动态的，其中ETS因子将以ETS-GATA-Ebox基序决定靶基因的特异性以及随后的细胞命运。AIM1将决定ER71在造血诱导中发挥作用的时间窗。AIM2将确定ER71位于ETS层次结构的顶端，并直接激活ETS基因的表达。AIM3将确定ER71介导的ETS激活导致ETS-GATA2-SCL靶基因的协同激活和造血程序的诱导。这一主题对造血和血管领域的发育生物学来说是最基本的。这项工作的成功完成将使人们更深入地了解胚胎发育过程中造血系统是如何建立的。这些知识也将直接适用于多能干细胞在再生生物学和医学、骨髓造血和造血疾病方面的研究。 与公共卫生相关：这项拨款建议是为了了解正在发育的小鼠胚胎中血液系统是如何建立的。为了实现这一目标，我们将鉴定ETS主调控子ER71及其下游靶基因。我们认为ER71可以诱导其他ETS基因，这些ETS基因随后与其他主要调控因子GATA2和SCL一起激活造血程序诱导。了解这些主要调节者激活的基因程序将是很重要的。这项研究的结果与基础研究、再生医学和血液疾病高度相关。

DESCRIPTION (provided by applicant): Our long-term goal is to understand how the hematopoietic system is established in the developing embryo. ER71, Fli1, Erg, Ets1, Ets2, Elk3 and Tel Ets transcription factors as well as GATA2 and Scl are relevant for hematopoietic and vascular development during embryogenesis. They are expressed in hematopoietic and endothelial cells. Moreover, mice display varying degrees of hematopoietic and vascular defects when any of these genes is inactivated. Clearly, there is an overlap in the gene regulatory network downstream of Ets, GATA2 and Scl that we need to understand. Indeed, emerging studies argue that Ets-GATA-Ebox motif is critical for hematopoietic development. For example, Runx1 expression in hematopoietic stem cells is regulated by the Ets-GATA-Ebox motif. As for Ets factors, it is not yet apparent how the Ets factor specificity is determined nor how much of the functional redundancy exists among Ets factors in hematopoietic and vascular development. We have exciting preliminary data suggesting that there is an Ets hierarchy and that ER71 is at the top. Notably, ER71 deficiency leads to the most severe hematopoietic and vascular defects compared to other Ets factors, GATA2 or Scl deficiency. Intriguingly, ER71 expression during embryogenesis is transient: it is expressed in the primitive streak and large vessels of dorsal aorta where definitive hematopoietic program is activated. In later stages yolk sac, fetal liver or bone marrow, ER71 expression is greatly reduced, but other Ets factor expression is prominent. From these data, we envision that ER71 induces Ets-GATA2-Scl triad mediated hematopoietic program activation. In this proposal, we test the following hypotheses: 1) ER71 is at the top of the hierarchy of Ets transcription factors in hematopoietic program induction; 2) Ets transcription factors together with GATA2 and Scl will activate primitive and definitive hematopoietic program; 3) Ets-GATA2-Scl triad is dynamic, in which Ets factors will dictate the specificity of target gene with Ets-GATA-Ebox motif and subsequent cellular fate. Aim1 will determine the window of time that ER71 functions in hematopoietic induction. Aim2 will establish that ER71 is at the top of Ets hierarchy and directly activates Ets gene expression. Aim3 will determine that ER71 mediated Ets activation results in cooperative Ets-GATA2-Scl target gene activation and hematopoietic program induction. This topic is most fundamental to the developmental biology of hematopoietic and vascular fields. Successful completion of the work will lead to a deeper understanding of how hematopoietic system is established during embryogenesis. Such knowledge will also be directly applicable for pluripotent stem cell research on regenerative biology and medicine, bone marrow hematopoiesis and hematopoietic disorders. PUBLIC HEALTH RELEVANCE: This grant proposal is to understand how blood system is established in the developing mouse embryo. Toward this goal, we will characterize an Ets master regulator ER71 and its downstream target genes. We believe that ER71 induces other Ets genes and that these Ets genes subsequently activate hematopoietic program induction together with other master regulators GATA2 and Scl. It will be important to understand genetic program activated by these master regulators. The outcome of this study is highly relevant to basic research, regenerative medicine and blood disorders.