Constructing gene regulatory networks for asymmetric brain development

描述（由申请人提供）：大脑的不对称发育是脊椎动物的一个保守特征，被认为通过避免功能重复来增加处理能力。对于大脑不对称发育，人们理解得最好的模型系统是斑马鱼。斑马鱼松果体复合体包括松果体器官和左侧松果体旁器官，后者支配左侧缰核。我们之前的工作已经确定了一种转录因子tbx2b，它是顶骨旁细胞特化和左侧迁移所必需的（Snelson等人，2008），但预计可能会有更多的转录因子参与这一过程。通过识别更多的基因和分子来指导顶骨旁器官的形成，对顶骨旁细胞如何从松果体细胞分离和独立分化的全面描述将会出现。在这里，我提出了两种互补但不同的方法来描述导致不对称顶骨旁神经元发育的基因调控回路。首先是表征和鉴定一种新的无旁尖细胞的突变体，图坦卡蒙国王，它是从最近在Gamse实验室进行的基因筛选中分离出来的。我将使用标准简单序列长度多态性（SSLP）基因组标记进行定位克隆，以确定图坦卡蒙国王的突变基因。我还将使用细胞命运图谱、谱系特异性标记、上位实验和细胞死亡/增殖分析进一步表征图坦卡蒙王表型，以确定图坦卡蒙王突变体缺乏顶旁器官的原因。其次，我将重点构建一个基因调控网络，通过鉴定顶果体和松果体发育所需的转录因子。这些目标的共同作用不仅将阐明将细胞分配给松果体与旁松果体命运的遗传途径，而且还将推进我们目前对神经元多样化、不对称细胞迁移和大脑中左右不对称的分子机制的理解。

DESCRIPTION (provided by applicant): Asymmetric development of the brain is a conserved feature of vertebrates, and is thought to increase processing capacity by avoiding duplication of function. The best understood model system for asymmetric brain development is the zebrafish. The zebrafish pineal complex includes the pineal organ and the left-sided parapineal organ, which innervates the left habenular nucleus. Our previous works has identified one transcription factor, tbx2b, that is required for parapineal cell specification and left-sided migration (Snelson et al., 2008), but expect that more transcription factors will likely be involved in this process. By identifying more genes and molecules that direct the formation of the parapineal organ, a comprehensive description of how parapineal cells segregate from pineal cells and independently differentiate will emerge. Here, I propose two complementary yet distinct approaches to delineate the gene regulatory circuitry that lead to the development of the asymmetric parapineal neurons. The first is to characterize and identify a novel parapineal-absent mutant, king tut, isolated from a recent genetic screen conducted in the Gamse laboratory. I will perform positional cloning with standard simple sequence length polymorphism (SSLP) genomic markers to identify the gene mutated in king tut. I will also further characterize the king tut phenotypes using cell fate mapping, lineage-specific markers, epistasis experiments, and cell death/proliferation assays to determine why king tut mutants lack a parapineal organ. Secondly, I will focus on constructing a gene regulatory network by identifying transcription factors required for parapineal and pineal development. These aims collectively will not only shed light on the genetic pathways that assign cells to a pineal versus a parapineal fate, but also advance our current understanding for the molecular mechanisms of neuronal diversification, asymmetrical cell migration and left-right asymmetry in the brain.)