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。这项拨款申请的重点是研究控制造血发育的感应信号和下游分子。利用胚胎干细胞（ES）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。在BMP、Notch和Wnt的抑制作用下，Ets转录因子Er71的表达明显下调。在ES细胞中增强ER71的表达导致FLK1+中胚层的强烈诱导，当被BMP、Notch和Wnt抑制阻断时，FLK1+中胚层的恢复生成，并增强造血细胞和内皮细胞的生成。此外，缺乏Er71的小鼠不能表达Flk1，在妊娠早期死亡，并表现出严重的血管缺陷，这与Flk1缺失小鼠表型高度相似。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、成血管细胞和造血发育的建立至关重要，而ER71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下游效应体。目的1是验证ER71在成血管细胞和造血系统的发生中起关键作用的假设。我们将在ER71-/- ES细胞和野生型胚胎之间产生并表征嵌合小鼠。我们也将产生和表征条件ER71基因敲除小鼠。目的2是验证ER71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。胚胎干细胞的体外分化，染色质免疫沉淀（ChIP）， ChIP- ChIP和ChIP-测序将用于鉴定和表征ER71靶基因。目的3是验证BMP信号在调节造血发育中与Wnt和/或Notch基因相互作用的假设。化合物、条件Smad4、nicastrin和2-catenin小鼠将被分析血液表型。我们认为，这些研究与造血发育的基本问题有关，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胚胎干细胞中产生造血干细胞和参与血液疾病的临床干预的最重要目标直接相关。公共卫生相关性：本拨款提案旨在定义调节血液形成的信号。具体来说，我们将研究BMP， Wnt和Notch在血液发育中的需求。我们还将验证一个假设，即ER71，一个Ets转录因子，整合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对血液疾病的干预至关重要。