HEMATOPOIETIC COMMITMENT: MOLECULAR MECHANISMS

造血承诺：分子机制

• 批准号: 7687336
• 负责人: KYUNGHEE CHOI
• 金额: $ 38万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-10 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7687336
• 关键词: Applications Grants; BHLH Protein; Blood; Blood Vessels; Bone Morphogenetic Proteins; Cells; Clinical; Defect; Development; Disease; Embryo; Endothelial Cells; Event; Future; Gene Targeting; Generations; Genetic; Goals; Grant; Hematopoiesis; Hematopoietic; Hematopoietic System; Hematopoietic stem cells; In Vitro; Intervention; Investigation; KDR gene; Knockout Mice; Knowledge; Mesoderm; Modeling; Molecular; Mus; Outcome; Outcome Study; Pathway interactions; Phenotype; Play; Pregnancy; Receptor Protein-Tyrosine Kinases; Regenerative Medicine; Role; Signal Transduction; Testing; Transcriptional Regulation; base; blastocyst; chromatin immunoprecipitation; embryonic stem cell; nicastrin protein; notch protein; progenitor; public health relevance; stem; transcription factor

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.

描述（由申请人提供）：我们的长期目标是了解造血系统是如何在发育中的胚胎中建立的。这项资助申请的重点是研究控制造血发育的诱导信号和下游分子。通过利用胚胎干细胞（ES细胞）的体外分化模型，我们鉴定了骨形态发生蛋白（BMP）、Wnt和Notch信号在Flk-1+中胚层形成和分化中起着不可或缺的作用。Ets转录因子Er71的表达被BMP、Notch和Wnt抑制剂显著下调。ES细胞中增强的ER 71表达导致FLK 1+中胚层的稳健诱导，当被BMP、Notch和Wnt抑制阻断时挽救FLK 1+中胚层的产生，并增强造血和内皮细胞的产生。此外，Er71缺陷的小鼠不能表达Flk1，在妊娠早期死亡，并显示出严重的血液和血管缺陷，这与Flk1缺失小鼠表型非常相似。基于这些初步发现，我们假设组合的BMP、Wnt和Notch信号对于Flk-1+中胚层、成血管细胞和造血发育的建立至关重要，并且ER 71是整合这些上游信号并调节Flk-1+中胚层、成血管细胞和造血发育的形成的下游效应物。目的1：验证ER 71在成血管细胞和造血系统发生中的重要作用。我们将产生和表征ER 71-/-ES细胞和野生型胚胎之间的嵌合小鼠。我们还将产生和表征条件性ER 71敲除小鼠。目的2是验证ER 71及其靶基因网络调控Flk-1+中胚层、成血管细胞和造血发育的假设。ES细胞的体外分化、染色质免疫沉淀（ChIP）、ChIP芯片和ChIP测序将用于鉴定和表征ER 71靶基因。目的3是检验BMP信号传导在调节造血发育中与Wnt和/或Notch遗传相互作用的假设。将分析化合物、条件性Smad4、nicastrin和2-连环蛋白小鼠的血液表型。我们相信，拟议的研究是相关的造血发育的基本问题，研究的结果将促进我们的理解调节造血发育的分子途径。从这些研究中获得的知识与从ES细胞产生造血干细胞的最重要目标和涉及血液病的临床干预直接相关。公共卫生相关性：这项拨款提案旨在定义调节血液形成的信号。具体而言，我们将研究BMP，Wnt和Notch在血液发育中的需求。我们还将测试一个假设，即ER 71，Ets转录因子，整合BMP，Wnt和Notch信号传导和调节血液形成。这一结果对未来利用胚胎干细胞的再生医学和血液病干预至关重要。