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始终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）。人脑的横截面揭示了精心隔离的中心，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左/右差异。例如，布罗卡区（一个编码语言的区域）在大多数右撇子、发育正常的个体的左侧较大（Fossi et al 2004）。不对称性并不是人类大脑所独有的：在整个脊椎动物进化枝中都可以看到大脑结构、基因表达和功能的侧向差异（Concha 和 Wilson 2001）。 我们发现分泌途径基因是斑马鱼不对称性形成的一个促成因素。该基因（sec6111）是脊椎动物易位子的主要亚基，易位子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）。我们怀疑毛孔本身是否具有神经发生能力；它可能调节不对称通路的早期组成部分。）发育遗传学代表了一种还原论学科，有可能帮助解码脊椎动物神经系统的奥秘。利用斑马鱼等模型生物，我们可以在不太复杂的环境中监测不对称的大脑发育，同时保留与人类同源基因的高度保守性。斑马鱼适用于功能获得和丧失技术，并具有强大的转基因监测能力。这使我们能够可视化有助于早期大脑发育的早期细胞行为、连接和运动，并仔细参考各个基因的贡献。 在我的研究中，我计划研究 sec6111 基因影响斑马鱼缰核神经发生的几种可能机制。我将首先检查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、通过 BrdU 掺入进行出生日期分析以及腹侧上丘脑祖细胞迁移的延时显微镜来区分。接下来，我将重点关注上丘脑顶板，这是发育中的神经系统中至关重要的信号中心。我们相信顶板影响缰核祖细胞的迁移，并且Notch信号家族可能调节顶板的形成。顶板标记的原位杂交和顶板的激光烧蚀阐明了与野生型相比，sec6111 和 Notch 途径突变体中这种背部结构的影响。最后，我们将通过遗传镶嵌分析、RNA 拯救和基因的缰核特异性表达来研究 sec6111 是否在缰核祖细胞或顶板中起作用。 公共卫生相关性：人脑的左右半球生来并不相同：大脑的某些区域单方面控制行为。这些左/右差异包括特殊功能，例如左半球特定的语言编码（De Fossi et al 2004），并且有人认为，不对称性在精神分裂症（Mitchell and Crow 2005）、自闭症（De Fossi et al 2004）和阅读障碍（Leonard and Eckert 2008）中受到破坏。通过表征斑马鱼中导致不对称性发展的高度保守基因，我们可以推断出它们的人类对应基因，以便更好地理解大脑中不对称性是如何以及为何产生的。

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.