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）：小鸡的听觉系统与哺乳动物的听觉系统相似，在过去的 40 年里一直是研究声音处理、神经回路发育和退化的宝贵研究工具，主要是因为它在整个发育过程中都具有可访问性。直到最近，在系统中进行基因操作的工具，无论是基因过表达还是基因敲除，都主要局限于早期胚胎时期。最近，基于转座子的载体已被证明可以在小鸡中表达。这些载体的优点是，当通过卵内电穿孔在 E2 处导入时，它们会整合到鸡基因组中，并且可以以时间方式进行调节，这意味着基因在 E2 处整合，但直到所需时间才表达。 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、大细胞核 (NM) 和板层核 (NL) 中具有有趣且独特的表达模式。 NL 是双齿神经元的层层，在背侧接收来自同侧 NM 轴突的兴奋性输入，在腹侧接收来自对侧 NM 过程的兴奋性输入。从突触发生开始，TrkB 表达在 NL 中增加，分离到腹侧树突，并在孵化期间保持在那里。在哺乳动物的中枢神经元中，TrkB 对于树突生长、树枝化和突触的维持至关重要。其中许多研究都是在培养的神经元上进行的，而在转基因动物的体内进行的研究要少得多，并且没有调节特定细胞表达的精细时间能力。此外，TrkB 有 2 种主要亚型，均存在于哺乳动物和雏鸡脑干中。我们建议在雏鸡中利用基于转座子的载体来调节发育特定时间的 TrkB 同工型表达，以剖析它们在细胞迁移、树突发生、突触发生以及 NM/NL 突触连接的细化和维护中的作用。 Sato 等人报道的 Tol2-强力霉素调节载体。 (2007) 将与 TrkB-shRNA-mir30 表达盒一起使用来调节 TrkB 同工型敲低。这里首创的技术，特定细胞中胚胎后期 shRNA 表达和 TrkB 基因表达的稳定整合和时间调控，对于其他研究鸡胚胎中后期发育过程的人来说将非常有用。公共健康相关性：小鸡的听觉回路与哺乳动物相似，更容易研究，但直到最近，小鸡还缺乏了解该系统发育和退化的分子基础的技术。我们将使用新开发的基因表达系统来暂时调节雏鸡听觉系统中特定细胞中的重要分子，以了解它们在发育和维持适当神经连接中的作用