The Vertebrate Translocon Sec61alpha is Required for Habenular Asymmetry

描述(由申请人提供)：对人脑的科学方法一直受到神经元数量的限制：估计有100亿到1万亿个神经元(Williams和Herrup，1998)。人类大脑的横切面揭示了精心分离的中心，这些中心可以调节精确的身体功能或复杂的情绪。两个半球的对比揭示了L/R的许多差异。例如，Broca区，一个编码语言的区域，在大多数惯用右手、正常发育的人的左侧更大(Fossi等人，2004年)。不对称并不是人脑独有的：在脊椎动物分支中可以观察到大脑结构、基因表达和功能的侧向差异(Concha和Wilson 2001)。我们已经暗示一个分泌途径基因是导致斑马鱼发育不对称性的一个因素。该基因，sec6111，是脊椎动物转位蛋白的主要亚基，内质网上的一个小孔，接受新翻译的多肽进入分泌途径(Rapoport 2007)。我们怀疑毛孔本身是否具有神经生成能力；它可能调节不对称通路的早期成分。)发育遗传学代表了一门简化论者的学科，有可能帮助破解脊椎动物神经系统的奥秘。利用斑马鱼这样的模式生物，我们可以在不那么复杂的环境中监测不对称的大脑发育，同时保持对人类同源物的高度保守的基因同源性。斑马鱼对功能的获得和丧失都具有可控性，并具有强大的转基因监测能力。这使我们能够通过仔细参考单个基因的贡献来可视化有助于早期大脑发育的早期细胞行为、连接和运动。在我的研究中，我计划研究sec6111基因影响斑马鱼缰核神经发生的几种可能机制。我将首先研究易位子对缰核亚核神经发生的影响，这可以通过特定标记基因的原位杂交、BrdU掺入的出生日期分析以及从腹侧上皮祖细胞迁移的时间推移显微镜来区分。接下来，我将重点介绍上皮层顶板，它是发育中的神经系统中的一个重要信号中心。我们认为，顶板影响缰核前体细胞的迁移，而Notch信号家族可能调节顶板的形成。与野生型相比，屋顶板标记的原位杂交和激光消融屋顶板阐明了这种背部结构在sec6111和Notch途径突变体中的影响。最后，我们将通过基因镶嵌分析、RNA拯救和缰核特异表达来研究sec6111是作用于缰系祖细胞还是作用于屋顶板。 与公共健康相关：人类大脑的左右半球并不是生来就相等的：大脑的某些区域单方面控制行为。L/R的这些差异包括特殊功能，例如语言的左半球特有编码(De Fossi等人，2004年)，并且有人认为精神分裂症(Mitchell和Crow，2005年)、自闭症(De Fossi等人，2004年)和阅读障碍(Leonard和Eckert，2008年)存在不对称现象。通过表征导致斑马鱼不对称性发展的高度保守的基因，我们可以推断出它们的人类同行，以更好地了解大脑如何以及为什么产生不对称性。

DESCRIPTION (provided by applicant): The scientific approach to the human brain has always been limited by the sheer number of neurons: estimated at 10 billion to 1 trillion neurons (Williams and Herrup 1998). Cross sections through a human brain reveal carefully segregated centers that can regulate precise bodily functions or complex emotions. A comparison between the hemispheres uncovers many L/R differences. For example, Broca's area, an area which codes for language, is larger on the left side of most right-handed, normally developed individuals (Fossi et al 2004). Asymmetry is not unique to the human brain: lateralized differences in brain architecture, gene expression, and function can be witnessed across the vertebrate clade (Concha and Wilson 2001). We have implicated a secretory pathway gene as a contributing factor to developing asymmetry in the zebrafish. This gene,)sec6111, is the major subunit of the vertebrate translocon, a pore on the endoplasmic reticulum which accepts newly translated peptides into the secretory pathway (Rapoport 2007). We doubt that the pore itself has neurogenic capacity; it likely regulates early components of the asymmetry pathway.) Developmental genetics represents one reductionist discipline with potential to help decode the mysteries of the vertebrate nervous system. Utilizing a model organism such as the zebrafish, we can monitor asymmetric brain development in a less intricate environment, yet retain high conservation of gene identity to human homologs. The zebrafish is amenable to both gain and loss of function techniques, and has powerful transgenic monitoring capacity. This allows us to visualize early cell behaviors, connections, and movements that contribute to early brain development, with careful reference to the contributions of individual genes. In my research, I plan to examine several possible mechanisms by which the sec6111 gene impacts habenular neurogenesis in the zebrafish. I will first examine the effects of the translocon on habenular subnuclear neurogenesis, which can be distinguished through in situ hybridization for specific marker genes, birth-date analysis through BrdU incorporation, and time-lapse microscopy of progenitor cell migration from the ventral epithalamus. Next, I will focus on the epithalamic roof plate, a crucial signaling center in the developing nervous system. We believe the roof plate influences habenular progenitor migration, and that the Notch signaling family may regulate roof plate formation. in situ hybridization for roof plate markers and laser ablation of the roof plate with illuminate the impact of this dorsal structure in both sec6111 and Notch pathway mutants, as compared to wild type. Finally, we will investigate whether sec6111 acts in the habenular progenitors or in the roof plate, through genetic mosaic analysis, RNA rescue, and habenular-specific expression of the gene. PUBLIC HEALTH RELEVANCE: The left and right hemispheres of the human brain are not created equal: some regions of the brain control behaviors unilaterally. These L/R differences include specialized functions, such as the left hemisphere-specific coding of language (De Fossi et al 2004), and it has been suggested that asymmetry is disrupted in schizophrenia (Mitchell and Crow 2005), autism (De Fossi et al 2004), and dyslexia (Leonard and Eckert 2008). By characterizing the highly conserved genes that contribute to the development of asymmetry in zebrafish, we can extrapolate to their human counterparts for a better understanding of how and why asymmetry is generated in the brain.