Mammalian genetic analysis of embryonic neural overgrow*

描述（由申请人提供）：本提案旨在完成一项正在进行的 绘制了小鼠中轴缺陷相关基因的图谱，并继续研究一种新的 克隆了斑马鱼的神经基因突变。一个最终目标是制作一个模型 它在老鼠。到目前为止，许多模式生物已被用于 分子发育研究：那些有很好的遗传学或 其发展阶段是容易获得的，但由于技术原因 基因研究不是一个选择。最近，斑马鱼已经开始使用 因为它同时满足这两个标准。它的优点之一是它是一个 脊椎动物，一天可以获得数百个胚胎，这些胚胎是 透明的，可以很容易地看到。因此，早期的新表型 神经发育是明显的，而它们在哺乳动物中是不可接近的。 在斑马鱼的逆转录病毒突变体筛选中，突变具有病毒标签， 从麻省理工学院的霍普金斯屏幕上，我选择了 分析一种隐性致死基因（Hi904），它会导致组织混乱和肿瘤样 神经系统的过度生长。该基因已被克隆，是新的。我们 将在鱼身上做一些很容易完成的实验，其余的在老鼠身上做。 在那里我们可以通过同源重组重建突变。实验 包括：胚胎中Hi904信息的表达研究， 使用充分表征的分子标记物进行分析，以评估 突变胚胎的神经系统，以及 分化这将通过原位杂交来实现， 抗体染色。在小鼠中的实验将包括：获得完整的 包括预期的可变剪接形式的小鼠cDNA， 正常组织和肿瘤组织以及胚胎中的原位杂交。我们将解决 通过将突变的鱼细胞移植到裸鼠体内来提高致瘤性。的中国梦作出 用于功能研究的抗体。小鼠中的突变将使用 同源重组Hi904的生化和细胞功能 蛋白质将通过研究其定位和相互作用来探索。是 预计这组基因将定义一种新的肿瘤抑制因子， 在儿童原始神经外胚层肿瘤的发生中的作用。

DESCRIPTION (provided by applicant): This proposal is to complete an ongoing mapping of genes involved in axis defects in mice and move on to study a new and cloned zebrafish neurogenic mutation. One ultimate goal is to make a model of it in mouse. To date, a number of model organisms have been used in the study of molecular development: those that have really good genetics or ones for which developing stages are readily accessible but for technical reasons genetic studies are not an option. Recently, zebrafish have come into use because it satisfies both criteria. Among its advantages are that it is a vertebrate, hundreds of embryos can be obtained in a day, the embryos are transparent and can be easily visualized. Thus, novel phenotypes in early neural development are evident whereas they would be inaccessible in a mammal. In a retroviral mutant screen in zebrafish, a mutation has a viral tag that can be used to easily clone it. From the Hopkins screen at MIT, I have chosen to analyze a recessive lethal (Hi904) that causes disorganization and tumor-like overgrowth of the nervous system. The gene has been cloned and is novel. We will do experiments that are easily accomplished in fish, and the rest in mice where we can recreate the mutation by homologous recombination. The experiments in zebrafish will include: expression studies of Hi904 message in embryos, analysis with well-characterized molecular markers to assess cell number in the nervous system of mutant embryos, and also patterning and state of differentiation. This will be accomplished by in situ hybridization and antibody staining. The experiments in mice will include: obtaining the complete mouse cDNAs including expected alternative splice forms, expression analysis in normal and tumor tissues and in situ hybridization in embryos. We will address tumorigenicity by transplanting mutant fish cells into nude mice. We will make antibody for functional studies. Mutations in mouse will be generated using homologous recombination. The biochemical and cellular function of Hi904 protein will be explored by studying its localization and interactions. It is expected that this set of genes will define a new tumor suppressor that plays-a role in the development of primitive neuroectodermal tumors of children.