The Vertebrate Translocon Sec61alpha is Required for Habenular Asymmetry

描述（由申请人提供）：研究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 and Herrup 1998）。人类大脑的横截面显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。两个半球之间的比较揭示了许多L/R差异。例如，对于大多数惯用右手、发育正常的人来说，负责语言编码的布洛卡区（Broca’s area）位于左侧较大（Fossi et al 2004）。不对称并非人类大脑所独有：在整个脊椎动物进化分支中都可以看到大脑结构、基因表达和功能的偏侧差异（Concha和Wilson 2001）。我们已经暗示分泌途径基因作为一个促进因素发展不对称斑马鱼。这个基因，sec6111，是脊椎动物转位的主要亚基，转位是内质网上的一个孔，它接受新翻译的肽进入分泌途径（Rapoport 2007）。我们怀疑毛孔本身具有神经生成能力；它可能调控着不对称通路的早期组成部分。)发育遗传学代表了一个简化主义的学科，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基因的高度保守性。斑马鱼对功能获得和功能丧失技术都具有适应性，具有强大的转基因监测能力。这使我们能够将早期细胞行为、连接和运动可视化，这些行为、连接和运动有助于早期大脑发育，并仔细参考个体基因的贡献。在我的研究中，我计划研究sec6111基因影响斑马鱼缰神经发生的几种可能机制。我将首先研究易位对habenular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，通过BrdU结合的出生日期分析和前体细胞从腹侧上皮迁移的延时显微镜来区分。接下来，我将重点介绍上皮细胞顶板，这是发育中的神经系统的重要信号中枢。我们认为顶板影响habenular祖细胞的迁移，Notch信号家族可能调控顶板的形成。与野生型相比，对顶板标记的原位杂交和顶板的激光消融阐明了sec6111和Notch途径突变体中这种背侧结构的影响。最后，我们将通过遗传镶嵌分析、RNA拯救和基因的habenular特异性表达来研究sec6111是在habenular祖细胞中还是在顶板中起作用。

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.