Role of Herp3 in Bmp Mediated Neurogenesis

Herp3 在 Bmp 介导的神经发生中的作用

• 批准号: 7112336
• 负责人: ALEXANDER G BASSUK
• 金额: $ 16.97万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-15 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7112336
• 关键词: RNA interference; biological signal transduction; bone morphogenetic proteins; cell differentiation; cell line; cell surface receptors; chromatin immunoprecipitation; developmental genetics; developmental neurobiology; embryo /fetus; embryogenesis; gel mobility shift assay; genetic regulation; genetically modified animals; immunocytochemistry; intermolecular interaction; laboratory mouse; nerve stem cell; neurogenesis; neurogenetics; protein structure function; transcription factor

DESCRIPTION (provided by applicant): The ability to manipulate neural progenitor cell differentiation may facilitate the treatment of many developmental, acquired and neurodegenerative processes, and understanding neuronal lineage determination may provide valuable insight into the mechanisms underlying neural physiology and disease. Lineage commitment by progenitor cells is influenced by epigenetic signals such as the bone morphogenetic proteins (Bmps) and the Delta/Notch lig and/receptor complexes that activate specific sets of transcriptional activators and repressors. Whereas many studies have focused on the signaling cascades regulating these distinct pathways, not much is known regarding how these pathways may interact to coordinately regulate neural stem cell fates. Recent evidence suggest that Bmp and Notch signaling may both activate members of the Hes (named after the Drosophila homologues Hairy and Enhancer of Split) and Hes-related (Herp) families of transcription factors, and that physical interactions among members of these families influence lineage fate. The protein Herp3 is a member of the Herp family of transcription factors whose function in neuronal development has yet to be elucidated. The goal of this research plan is to explore the role of the Herp3 protein in neuronal cell fate determination in response to Bmp signaling. The primary hypothesis of the proposed research is that Herp3 is involved in Bmp-mediated neuronal differentiation, and that interactions between Herp3 and Hes family members mediate Herp3 function. The Specific Aims of the proposed project are: 1) to identify the transcriptional targets of Herp3 and its role in neuronogenesis; 2) to analyze the function of the Herp3-Hes interaction; 3) to define the functional importance of Herp3 expression in response to Bmp signaling in vivo. Experimental design and methods to accomplish the first Aim include RNA inhibition (RNAi), transient transfection, and the chromatin precipitation (CHIP) cloning assay to identify target regulatory elements of Herp3 transcriptional regulation. The second Aim will involve electophoretic mobility shift assays (EMSAs) and transient transfections to evaluate the role of the Hes-Herp3 interaction on DNA binding. The third Aim will involve in situ hybridization and immunohistochemistry in normal mice and in mice with mutations of the Bmp receptor to examine normal Herp3 expression in vivo and Herp3 expression in response to Bmp signaling. The third Aim will also involve viral mis-expression of Herp3 and of RNAi molecules of Herp3, to evaluate the in vivo function of the Herp3 protein. The experiments described in this research plan should allow a detailed account of the basic function of the Herp3 protein in neuronal lineage determination in response to Bmp signaling, thereby elucidating the mechanisms by which neuronal differentiation is deranged in neurological disease.

描述（由申请人提供）：操纵神经祖细胞分化的能力可以促进许多发育、后天和神经退行性过程的治疗，并且理解神经元谱系决定可以为神经生理学和疾病的潜在机制提供有价值的见解。祖细胞的谱系定型受到表观遗传信号的影响，例如骨形态发生蛋白 (Bmps) 和激活特定转录激活因子和阻遏因子组的 Delta/Notch lig 和/受体复合物。尽管许多研究都集中在调节这些不同途径的信号级联上，但对于这些途径如何相互作用以协调调节神经干细胞的命运知之甚少。最近的证据表明，Bmp 和 Notch 信号传导可能都会激活 Hes（以果蝇同源物 Hairy 和 Split 增强子命名）和 Hes 相关 (Herp) 转录因子家族的成员，并且这些家族成员之间的物理相互作用会影响谱系命运。 Herp3 蛋白是转录因子 Herp 家族的成员，其在神经元发育中的功能尚未阐明。该研究计划的目标是探索 Herp3 蛋白在响应 Bmp 信号传导的神经元细胞命运决定中的作用。本研究的主要假设是 Herp3 参与 Bmp 介导的神经元分化，并且 Herp3 和 Hes 家族成员之间的相互作用介导 Herp3 功能。该项目的具体目标是： 1）确定 Herp3 的转录靶标及其在神经元发生中的作用； 2) 分析Herp3-Hes相互作用的功能； 3) 定义 Herp3 表达响应体内 Bmp 信号传导的功能重要性。实现第一个目标的实验设计和方法包括 RNA 抑制 (RNAi)、瞬时转染和染色质沉淀 (CHIP) 克隆测定，以鉴定 Herp3 转录调控的靶调控元件。第二个目标将涉及电泳迁移率变动分析 (EMSA) 和瞬时转染，以评估 Hes-Herp3 相互作用对 DNA 结合的作用。第三个目标将涉及正常小鼠和 Bmp 受体突变小鼠的原位杂交和免疫组织化学，以检查体内正常 Herp3 表达以及响应 Bmp 信号传导的 Herp3 表达。第三个目标还将涉及 Herp3 和 Herp3 RNAi 分子的病毒错误表达，以评估 Herp3 蛋白的体内功能。本研究计划中描述的实验应该能够详细说明 Herp3 蛋白在响应 Bmp 信号传导的神经元谱系测定中的基本功能，从而阐明神经系统疾病中神经元分化紊乱的机制。