The Role of Zic1 in Neural Patterning and in Mid-/hindbrain Boundary Formation

1)智力价值：神经系统的正确形成对于任何有活力的多细胞生物的发育都是至关重要的。神经管的最初形成，随后分化为中枢神经系统的各个隔室，是一个高度协调的过程。这个过程是由复杂的和动态的基因表达模式直接在分子水平。这些基因在发育的早期就开始表达，那时将形成神经系统的细胞刚刚被搁置一边。由此产生的基因表达模式指明了大脑的不同部分，如前脑、中脑和后脑。中脑和后脑之间的连接处，即中脑/后脑边界（MHB），对于提供中脑（视顶盖）和后脑前侧（小脑）正确发育的信号至关重要。当这些MHB信号在胚胎的不同区域（例如脊髓区域）错误表达时，它们将新环境改变的命运导向中脑/后脑特征。MHB在原肠期开始形成，并且发现了一个日益复杂的基因调控网络来调节其形成。有一个基因在神经系统发育的早期就很活跃，它就是转录因子zic1。zic1参与了神经板的早期分子模式，初步数据表明它参与了MHB的形成。因此，本研究的目标是双重的：首先，确定zic1的表达是否为早期神经发育所必需，其次，确定zic1是否作为中脑/后脑边界形成的上游调节剂。这些研究将在爪蟾身上进行，因为它的早期发育非常容易进行实验。在非洲爪蟾胚胎中会表达多种显性干扰结构，以破坏正常的zic1功能。这些实验将确定zic1是否是神经模式所必需的，它是否作为转录的激活和/或抑制因子，以及它在MHB形成中起什么作用。为了进一步剖析产生MHB的分子相互作用以及zic1在这些相互作用中的参与，将使用外胚层外植体中的基因表达（动物帽测定）。这些外植体将模仿MHB在一个更容易控制的组织中形成的各个方面，远离正常发育过程中的各种信号。总之，来自这些实验的数据将显著增强我们对驱动神经系统不同部分建立的分子机制的理解。2)更广泛的影响：拟议的研究活动将增加我们对大脑如何发育以及zic1如何促进MHB形成的理解。Zic1在发育过程中也有其他功能，并且在生物体中高度保守。因此，了解zic1如何适应爪蟾中产生MHB的分子相互作用，将有助于比较zic1的其他发育功能和其他生物的进化保护方面的共享和不同的调节途径。此外，拟议的研究活动将是私人调查计划的组成部分的教育活动。这项工作将由两名博士生完成。多个较小的项目将允许几个本科生参与独立的项目。私家侦探致力于美国原住民学生的教育，有两名美国原住民学生参与了她实验室的研究。其中一名美国原住民学生将参与这个项目和她在P.I.的工作去年夏天，他的实验室在全国SACNES会议上获得了杰出海报奖。其他美国原住民本科生很可能也会参与这项拟议的研究。此外，关于这项研究的讨论将成为pi教授的本科生和研究生讲座和实验课程的一部分。最后，这项研究将在蒙大拿州立大学进行，这是一个EPSCoR机构，并将为一些优秀的本科生提供重要的研究经验，否则他们可能没有机会参与基于发现的研究。

Christa MerzdorfMontana State UniversityProject summary 1) Intellectual merit: The correct formation of the nervous system is critical to the development of any viable multicelular organism. The initial formation of the neural tube, followed by its differentiation into the various compartments of the central nervous system, is a highly orchestrated process. This process is directed at the molecular level by complex and dynamic patterns of gene expression. These genes are expressed very early in development, when the cells that will form the nervous system are just being set aside. The resulting gene expression patterns specify the different parts of the brain, such as forebrain, midbrain, and hindbrain. The junction between the midbrain and the hindbrain, the midbrain/hindbrain boundary (MHB), is critical for providing the signals that allow correct development of the midbrain (optic tectum) and the anterior hindbrain (cerebellum). When these MHB signals are misexpressed in a different region of the embryo (for example the spinal cord region), they direct their new surroundings change fate to midbrain/hindbrain character. The MHB begins to form during gastrula stages and an increasingly complex gene regulatory network is found to regulate its formation. One gene that is active very early in the development of the nervous system is the transcription factor zic1. zic1 contributes to early molecular patterning of the neural plate and preliminary data indicate that it participates in formation of the MHB. Thus, the goals of this proposal are twofold: first, to determine whether zic1 expression is required for early neural development, and, second, to determine whether zic1 functions as an upstream regulator of midbrain/hindbrain boundary formation. These studies will be conducted in Xenopus, since its early development is highly accessible to experimentation. Various dominant interfering constructs will be expressed in Xenopus embryos to ablate normal zic1 function. These experiments will determine wether zic1 is required for neural patterning, whether it functions as an activator and/or repressor of transcription, and what role it plays in MHB formation. In order to dissect further the molecular interactions that give rise to the MHB and the involvement of zic1 in these interactions, gene expression in ectodermal explants (animal cap assays) will be used. These explants will mimic aspects of MHB formation in a more easily controlled tissue away from the various signals during normal development. Together, the data from these experiments will significantly enhance our understanding of the molecular mechanisms that drive establishment of the different parts of the developing nervous system.2) Broader impact: The proposed research activities will increase our understanding of how the brain develops and how zic1 contributes to MHB formation. zic1 performs other functions in development as well and is highly conserved among organisms. Thus, knowledge of how zic1 fits into the molecular interactions that give rise to the MHB in Xenopus will allow comparison of shared and divergent regulatory pathways with respect to other developmental functions of zic1 and with respect to evolutionary conservation in other organisms. In addition, the proposed research activities will be integral part of the P.I.'s educational activities. The work will be performed by two Ph.D. students. Multiple smaller projects will allow the involvement of several undergraduate students with independent projects. The P.I. is committed to the education of Native American students and has two Native American students involved in research in her laboratory. One of these Native American students will be participating in this project and her work in the P.I.'s laboratory was recognized for outstanding poster at the national SACNES conference last summer. It is likely that other Native American undergraduate students will be involved in the proposed research. Further, discussion of this research will be part of undergraduate and graduate lecture and laboratory courses taught by the P.I. Finally, this research will be performed at Montana State Universiry, which is an EPSCoR institution, and will provide critical research experience to a number of excellent undergraduate students who might otherwise not have the opportunity to be involved in discovery-based research.