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如何在发育中的胚胎中建立造血系统。该赠款应用的重点是对控制造血发育的归纳信号和下游分子的研究。通过利用胚胎茎（ES）细胞的体外分化模型，我们确定了骨形态发生蛋白（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。 ETS转录因子ER71的表达大大下降了BMP，Notch和Wnt抑制。 ES细胞中强制执行的ER71表达导致了FLK1+中胚层的强烈诱导，当通过BMP，Notch和Wnt抑制阻断时，拯救了FLK1+中胚层的产生，并增强了造血和内皮细胞的产生。此外，缺乏ER71的小鼠无法表达FLK1，死于早期妊娠，表现出严重的血液和血管缺陷，这些缺陷高度让人联想到FLK1 NULL小鼠表型。 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.目的1是检验一个假设，即ER71对于血管细胞和造血系统的起源至关重要。我们将在ER71 - /ES细胞和野生型胚胎之间生成和表征嵌合小鼠。我们还将生成并表征有条件的ER71敲除小鼠。目的2是检验一个假设，即ER71及其靶基因的网络调节FLK-1+中胚层，血管细胞和造血性发育。 ES细胞，染色质免疫沉淀（CHIP），芯片芯片和芯片序列的体外分化将用于识别和表征ER71靶基因。 AIM 3是检验一个假设，即BMP信号在调节造血发育时与Wnt和/或缺口遗传相互作用。将分析化合物，条件SMAD4，尼加斯特林和2-catenin小鼠的血液表型。我们认为，拟议的研究与发育性造血的基本问题有关，研究结果将提高我们对调节造血发展的分子途径的理解。从这些研究中获得的知识直接与来自ES细胞产生造血干细胞以及参与血液学疾病的临床干预措施的最大重要目标有关。公共卫生相关性：该赠款提案是为了定义调节血液形成的信号。具体来说，我们将研究血液发育中的BMP，WNT和Notch要求。我们还将检验一个假设，即ETS转录因子ER71整合了BMP，Wnt和Notch信号传导并调节血液形成。该结果对于利用胚胎干细胞和有关血液学疾病的干预措施的未来再生医学至关重要。