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

描述（申请人提供）：小鸡的听觉系统类似于哺乳动物的听觉系统，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重要研究工具，主要是因为它在整个发育过程中都是可获得的。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低，都主要局限于胚胎早期。最近，以转座子为基础的载体已被证明在鸡中表达。这些载体的优点是，当它们在E2时通过卵内电穿孔引入时，它们可以整合到小鸡基因组中，并且可以以一种时间方式进行调节，这意味着基因在E2时被整合，但直到所需的时间才表达。TrkB是结合神经营养因子BDNF的生长因子受体，在脑干听核大细胞核（NM）和层状核（NL）中有一个有趣而独特的表达模式。NL是双齿神经元的层流层，接受来自同侧NM轴突的背侧和来自对侧NM突的腹侧兴奋性输入。从突触发生开始，TrkB在NL中的表达增加，分离到腹侧树突，并在孵化过程中保持在那里。在哺乳动物的中枢神经元中，TrkB对树突生长、树突化和突触的维持至关重要。许多这样的研究都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有两种主要的同工型，它们都存在于哺乳动物和鸡的脑干中。我们建议利用小鸡中基于转座子的载体在发育的特定时期调节TrkB异构体的表达，以解剖它们在细胞迁移、树突发生、突触发生以及NM/NL突触连接的完善和维持中的作用。Sato等人（2007）报道的tol2 -强力霉素调节载体将与TrkB- shrna -mir30表达盒一起使用，以调节TrkB异构体的敲低。在这里开创的技术，shRNA和TrkB基因表达在特定细胞胚胎后期的稳定整合和时间调控将对其他研究鸡胚胎中后期发育过程的人有很大的帮助。

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