Focal and temporal regulation of TrkB gene expression in chick auditory brainstem

鸡听觉脑干 TrkB 基因表达的局灶性和时间性调节

基本信息

批准号：
8091892
负责人：
MARK ALLEN BOTHWELL
金额：
$ 23.92万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-05-01 至 2013-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8091892
关键词：
Animals Antibodies Architecture Auditory Auditory system Axon Brain Stem Brain-Derived Neurotrophic Factor Cell Death Cell Nucleus Cells Chick Embryo Complex Contralateral Data Deafferentation procedure Dendrites Development Developmental Process Dorsal Doxycycline Dyes Electroporation Embryo Event Exhibits Gene Expression Genes Genome Growth Growth Factor Receptors Hour Immunohistochemistry Injection of therapeutic agent Ipsilateral Length Maintenance Mammals Maps Molecular Molecular Profiling Mus Nervous System Part Neurons Neurotrophic Tyrosine Kinase Receptor Type 2 Pattern Pharmaceutical Preparations Phase Phosphotransferases Plasmids Play Process Protein Isoforms Publications RNA Splicing Receptor Protein-Tyrosine Kinases Regulation Relative (related person)Reporting Research Research Personnel Role Staging Synapses Synaptophysin System Techniques Time Transgenic Animals Trees Vertebrates Western Blotting Zebrafish auditory nuclei base cell motility genetic manipulation hatching in vivo interest neural circuit neurodevelopment neurotrophic factor novel overexpression protein expression receptor receptor binding relating to nervous system research study response small hairpin RNA sound spatiotemporal synaptogenesis tool trafficking vector

项目摘要

DESCRIPTION (provided by applicant): The chick auditory system is similar to that in mammals and has served as a valuable research tool in the study of sound processing, neural circuit development and degeneration for the past 40 years, mainly because of its accessibility throughout development. Until recently the tools to make genetic manipulations in the system, either gene overexpression or knockdown, have been limited mostly to early embryonic times. Recently transposon-based vectors have been shown to express in chick. The advantage of these vectors is that they integrate into the chick genome when introduced by in ovo electroporation at E2 and can be regulated in a temporal manner, meaning the genes are integrated at E2 but not expressed until the desired time. TrkB, the growth factor receptor that binds the neurotrophin BDNF, has an interesting and unique expression pattern in the two brainstem auditory nuclei, nucleus magnocellularis (NM) and nucleus laminaris (NL). NL is a laminar layer of bidentate neurons receiving excitatory input dorsally from ipsilateral NM axons, and ventrally from contralateral NM processes. From the start of synaptogenesis, TrkB expression increases in NL, segregates to the ventral dendrites, and remains there through hatching. In central neurons in mammals TrkB is crucial for dendrite growth, arborization and maintenance of synapses. Many of these studies have been done on neurons in culture, far fewer in vivo in transgenic animals, and without the fine temporal ability to regulate expression in specific cells. In addition, there are 2 major isoforms of TrkB and both are present in mammals and in the chick brainstem. We propose to exploit the use of transposon-based vectors in the chick to regulate TrkB isoform expression at specific times in development to dissect their roles in cell migration, dendritogenesis, synaptogenesis, and the refinement and maintenance of NM/NL synaptic connections. The Tol2-doxycycline regulated vectors reported by Sato et al. (2007) will be used with a TrkB-shRNA-mir30 expression cassette to regulate TrkB isoform knockdown. The techniques pioneered here, stable integration and temporal regulation of shRNA expression and TrkB gene expression at later embryonic stages in specific cells will be of great use others who study mid to late developmental processes in chick embryos. PUBLIC HEALTH RELEVANCE: The auditory circuit of the chick is similar to that in mammals and much more accessible to study, yet until recently the techniques to understand the molecular basis of development and degeneration of the system have been lacking in the chick. We will use a newly developed gene expression system to temporally regulate important molecules in specific cells in the chick auditory system to access their roles in the development and maintenance of proper neural connections

描述（由申请人提供）：鸡听觉系统与哺乳动物中的相似，并且在过去40年来研究声音处理，神经回路发展和退化的研究中，主要是因为它在整个开发过程中的可及性。直到最近，在基因过表达或敲低的基因过表达或敲低的系统中制造遗传操作的工具主要限于早期的胚胎时代。最近，基于转座子的矢量已显示出在雏鸡中表达。这些向量的优点是，当通过E2处的OVO电穿孔引入时，它们会整合到雏鸡基因组中，并且可以以时间方式调节，这意味着这些基因在E2上集成，但直到所需的时间才表达。 TRKB是结合神经营养蛋白BDNF的生长因子受体，在两个脑干听觉核，核细胞核（NM）（NM）和laminaris（NL）中具有有趣而独特的表达模式。 NL是从同侧NM轴突背侧接收兴奋性输入的双齿神经元的层层层，并从对侧NM过程进行腹侧。从突触发生的开始，TRKB表达在NL中增加，分离到腹侧树突，并通过孵化。在哺乳动物中的中央神经元中，TRKB对于突触的树突生长，树博和维持至关重要。这些研究中的许多研究都是针对培养的神经元进行的，在转基因动物中体内少得多，并且没有调节特定细胞表达表达的良好时间能力。此外，TRKB有2种主要的同工型，并且两者都存在于哺乳动物和鸡脑干中。我们建议利用基于转座子的载体在雏鸡中的使用来调节发育中特定时间的TRKB同工型表达，以剖析其在细胞迁移，树突生成，突触发生以及NM/NL突触连接的细化和维持中的作用。 Sato等人报告的TOL2-二氧环素调节载体。（2007年）将与TRKB-SHRNA-MIR30表达盒一起使用，以调节TRKB同工型敲低。此处开创的技术，在特定细胞中以后的胚胎阶段的shRNA表达和TRKB基因表达的稳定整合和时间调节将大大使用其他研究雏鸡胚胎中中期到晚发育过程的其他人。公共卫生相关性：小鸡的听觉电路与哺乳动物相似，并且更容易学习，但是直到最近，小鸡缺乏了解该系统发展和变性的分子基础的技术。我们将使用新开发的基因表达系统在雏鸡听觉系统中特定细胞中的重要分子来访问其在适当的神经连接的开发和维持中的作用