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。这项资助申请侧重于研究控制造血发育的诱导信号和下游分子。利用胚胎干细胞的体外分化模型，我们发现骨形态发生蛋白(BMP)、Wnt和Notch信号在Flk-1+中胚层的形成和分化中起着不可或缺的作用。抑制BMP、Notch和Wnt可显著下调ETS转录因子Er71的表达。在ES细胞中增强ER71的表达可强烈诱导Flk1+中胚层，当被BMP、Notch和Wnt抑制时挽救了Flk1+中胚层的生成，并促进了造血细胞和内皮细胞的生成。此外，Er71基因缺陷的小鼠无法表达Flk1，在怀孕早期死亡，并表现出严重的血管缺陷，这与Flk1缺失的小鼠表型高度相似。基于这些初步发现，我们假设BMP、Wnt和Notch信号的结合对Flk-1+中胚层、血管母细胞和造血发育的建立至关重要，而ER71是一个下游效应因子，整合了这些上游信号并调节Flk-1+中胚层、血管母细胞和造血发育的形成。目的1验证ER71在血管母细胞和造血系统的发生中起关键作用的假说。我们将创造和鉴定ER71-/-ES细胞和野生型胚胎之间的嵌合小鼠。我们还将培育和鉴定条件性ER71基因敲除小鼠。目的2验证ER71及其靶基因网络调控Flk-1+中胚层、血管母细胞和造血发育的假说。我们将利用ES细胞的体外分化、染色质免疫沉淀(CHIP)、CHIP-CHIP和芯片测序等方法来鉴定和鉴定ER71靶基因。目的3是验证一种假说，即BMP信号在基因上与Wnt和/或Notch在调节造血发育中相互作用。将分析复合、条件性Smad4、尼卡斯特林和2-连环素小鼠的血型。我们相信，所提出的研究与发育造血的基本问题有关，研究结果将促进我们对调节造血发育的分子途径的理解。从这些研究中获得的知识与从ES细胞生成造血干细胞这一最重要的目标和涉及血液疾病的临床干预直接相关。与公共健康相关：这项拨款提案是为了定义调节血液形成的信号。具体地说，我们将调查血液发育中对BMP、Wnt和Notch的需求。我们还将检验一种假说，即ER71是一种ETS转录因子，它整合了BMP、Wnt和Notch信号，并调节血液形成。这一结果将对未来利用胚胎干细胞的再生医学和有关血液病的干预措施至关重要。

DESCRIPTION (provided by applicant): Our long-term goal is to understand how the hematopoietic system is established in the developing embryo. This grant application focuses on the investigation of inductive signals and downstream molecules that control hematopoietic development. By utilizing the in vitro differentiation model of embryonic stem (ES) cells, we identified that bone morphogenetic protein (BMP), Wnt and Notch signals play an integral role in Flk-1+ mesoderm formation and differentiation. Expression of Er71, an Ets transcription factor, was greatly down regulated by BMP, Notch and Wnt inhibition. Enforced ER71 expression in ES cells resulted in a robust induction of FLK1+ mesoderm, rescued generation of FLK1+ mesoderm when blocked by BMP, Notch and Wnt inhibition, and enhanced hematopoietic and endothelial cell generation. Moreover, mice deficient in Er71 failed to express Flk1, died in early gestation and displayed severe blood and vessel defects that are highly reminiscent of the Flk1 null mouse phenotype. Based on these preliminary findings, we hypothesize that combined BMP, Wnt, and Notch signals are critical for the establishment of the Flk-1+ mesoderm, hemangioblast and hematopoietic development and that ER71 is a downstream effector that integrates these upstream signals and regulates the formation of Flk-1+ mesoderm, hemangioblast and hematopoietic development. Aim 1 is to test a hypothesis that ER71 is critical for the genesis of hemangioblast and hematopoietic system. We will generate and characterize chimeric mice between ER71-/- ES cells and wild type embryos. We will also generate and characterize conditional ER71 knockout mice. Aim 2 is to test a hypothesis that a network of ER71 and its target genes regulates Flk-1+ mesoderm, hemangioblast and hematopoietic development. In vitro differentiation of ES cells, chromatin immunoprecipitation (ChIP), ChIP-chip and ChIP-sequencing will be utilized to identify and characterize ER71 target genes. Aim 3 is to test a hypothesis that BMP signaling genetically interacts with Wnt and/or Notch in regulating hematopoietic development. Compound, conditional Smad4, nicastrin, and 2-catenin mice will be analyzed for blood phenotype. We believe that the proposed studies are pertinent to fundamental issues of developmental hematopoiesis and that the outcome of the studies will advance our understanding of the molecular pathways regulating hematopoietic development. The knowledge gained from these studies is directly relevant for the utmost important goal of generating hematopoietic stem cells from ES cells and clinical interventions involved in hematologic disorders. PUBLIC HEALTH RELEVANCE: This grant proposal is to define signals that regulate blood formation. Specifically, we will investigate BMP, Wnt and Notch requirements in blood development. We will also test a hypothesis that ER71, an Ets transcription factor, integrates BMP, Wnt and Notch signaling and regulates blood formation. The outcome will be critical for future regenerative medicine utilizing embryonic stem cells and interventions concerning hematologic disorders.