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

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，小鸡的听觉系统一直是研究声音处理、神经回路发育和退化的有价值的研究工具，这主要是因为小鸡的听觉系统在整个发育过程中都是可接近的。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减，都主要限于早期胚胎时期。最近转座子载体已被证明在鸡中表达。这些载体的优点是，当在E2通过卵内电穿孔引入时，它们整合到鸡基因组中，并且可以以时间方式调节，这意味着基因在E2整合，但直到所需的时间才表达。TrkB是与神经营养因子BDNF结合的生长因子受体，在脑干的两个听觉核团（大细胞核（NM）和板状核（NL））中具有有趣且独特的表达模式。NL是一个层状的双齿神经元，从同侧NM轴突背侧接受兴奋性输入，从对侧NM过程腹侧接受兴奋性输入。从突触发生开始，TrkB表达增加NL，分离到腹侧树突，并通过孵化保持在那里。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。这些研究中有许多是在培养的神经元上进行的，在转基因动物体内进行的研究要少得多，而且没有在特定细胞中调节表达的精细时间能力。此外，TrkB有2种主要的同种型，两者都存在于哺乳动物和鸡脑干中。我们建议利用转座子为基础的载体在小鸡调节TrkB亚型的表达在特定的时间在发展中剖析他们的作用，细胞迁移，树突状细胞，突触，和细化和维护NM/NL突触连接。由Sato等人（2007）报道的Tol 2-强力霉素调节的载体将与TrkB-shRNA-mir 30表达盒一起使用以调节TrkB同种型敲低。在此开创的技术，稳定的整合和时间调控的shRNA表达和TrkB基因表达在胚胎后期的特定细胞将是非常有用的其他人谁研究中晚期发育过程中的鸡胚。 公共卫生相关性：小鸡的听觉回路与哺乳动物的听觉回路相似，更容易研究，但直到最近，在小鸡中一直缺乏了解该系统发育和退化的分子基础的技术。我们将使用一个新开发的基因表达系统来暂时调节鸡听觉系统中特定细胞中的重要分子，以评估它们在正常神经连接的发育和维持中的作用

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