Constructing gene regulatory networks for asymmetric brain development

描述（由申请人提供）：脑的不对称发育是脊椎动物的保守特征，并且被认为通过避免功能的重复来增加处理能力。最好理解的不对称大脑发育模型系统是斑马鱼。斑马鱼松果体复合体包括松果体器和左侧松果体旁器，左侧松果体旁器支配左侧缰核。我们先前的工作已经鉴定了一种转录因子tbx 2b，其是会阴旁细胞特化和左侧迁移所需的（Snelson et al.，2008年），但预计更多的转录因子可能会参与这一过程。通过鉴定更多的基因和分子，指导松果体旁器官的形成，一个全面的描述如何松果体旁细胞分离松果体细胞和独立分化将出现。在这里，我提出了两个互补的，但不同的方法来描绘的基因调控电路，导致发展的不对称的松果体旁神经元。第一个是表征和确定一种新的旁松果体缺失突变体，图坦卡蒙，从最近的基因筛选在Gamse实验室进行分离。我将用标准的简单序列长度多态性基因组标记进行定位克隆来鉴定图坦卡蒙的突变基因。我还将进一步表征图坦卡蒙表型使用细胞命运映射，谱系特异性标记，上位性实验，细胞死亡/增殖测定，以确定为什么图坦卡蒙突变体缺乏会阴旁器官。其次，我将专注于构建一个基因调控网络，通过识别转录因子所需的松果体和松果体发育。这些目标不仅将揭示将细胞分配给松果体与松果体旁命运的遗传途径，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。 公共卫生相关性：人类大脑左右半球的专门化对于维持其正常功能至关重要。大脑偏侧性的缺失或破坏与各种发育性神经疾病有关，包括精神分裂症、自闭症和阅读障碍。拟议的实验利用一个强大的遗传系统，斑马鱼背侧间脑，以提高我们的知识，左右差异如何出现在发育中的大脑，通过表征大脑不对称的分子基础在一个非常简单的模型，神经元的松果体器官。

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.) PUBLIC HEALTH RELEVANCE: Specialization of the left and right hemispheres of the human brain is essential for sustaining its normal functions. Abnormalities or disruptions in brain laterality have been associated with a variety of developmental neurological conditions, including schizophrenia, autism, and dyslexia. The proposed experiments utilize a powerful genetic system, the zebrafish dorsal diencephalon, to enhance our knowledge of how left-right differences arise in the developing brain by characterizing the molecular basis of brain asymmetry in a very simple model, the neurons of the parapineal organ.