Constructing gene regulatory networks for asymmetric brain development

描述(申请人提供)：大脑的不对称发育是脊椎动物的保守特征，被认为通过避免功能重复来提高处理能力。对大脑发育不对称最了解的模型系统是斑马鱼。斑马鱼松果体复合体包括松果体和左侧松果体，左侧松果体支配左侧缰核。我们以前的工作已经确定了一种转录因子tbx2b，它是松果体细胞规范和左侧迁移所必需的(Snelson等人，2008年)，但预计可能会有更多的转录因子参与这一过程。通过识别更多指导松果体器官形成的基因和分子，将出现关于松果体细胞如何从松果体细胞分离并独立分化的全面描述。在这里，我提出了两种互补但不同的方法来描绘导致非对称松果体神经元发育的基因调控电路。第一个是从甘斯实验室最近进行的基因筛查中分离出一种新型的无松果体突变体King tut，并对其进行表征和鉴定。我将用标准的简单序列长度多态(SSLP)基因组标记进行定位克隆，以鉴定国王图坦卡蒙突变的基因。我还将通过细胞命运图谱、谱系特异性标记、上位性实验和细胞死亡/增殖分析进一步表征图坦卡蒙国王的表型，以确定为什么图坦卡蒙国王突变体没有松果体器官。其次，我将专注于通过识别松果体和松果体发育所需的转录因子来构建基因调控网络。这些目标的共同作用不仅将阐明将细胞分配给松果体而不是松果体命运的遗传路径，而且还将促进我们目前对大脑中神经元多样化、不对称细胞迁移和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.)