Mechanisms of axon guidance during development

发育过程中轴突引导的机制

• 批准号: 8557036
• 负责人: edward giniger
• 金额: $ 97.35万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8557036
• 关键词: Actins; Alternative Splicing; Animals; Axon; Biological Assay; Biosensor; Cell physiology; Cells; Collaborations; DNA Binding; Data; Dendrites; Development; Dominant-Negative Mutation; Drosophila genus; Embryo; Epithelial; Equilibrium; Fluorescence Resonance Energy Transfer; Genes; Genetic; Gleevec; Goals; Growth; Growth Cones; Guanosine Triphosphate Phosphohydrolases; Image; Individual; Life; Mammalian Cell; Modeling; Molecular; Molecular Genetics; Molecular Profiling; Monitor; Morphogenesis; Morphology; Mutation; Nature; Nerve; Neurodegenerative Disorders; Neurons; Ontology; Open Reading Frames; Output; Pathway interactions; Pattern; Phase; Phenotype; Phosphotransferases; Process; Protein Isoforms; Protein Tyrosine Kinase; Proteins; Publishing; Reagent; Regulation; Reporter; Reporting; Research; Role; Signal Pathway; Signal Transduction; Specificity; Structure; Surface; System; Testing; Vertebrates; Zinc Fingers; axon growth; axon guidance; base; cell motility; fly; gene function; genome-wide; in vivo; interest; kinase inhibitor; mutant; neurodevelopment; notch protein; programs; receptor; relating to nervous system; research study; transcription factor

We seek to answer two questions: how do neurons become connected during development, and why do they become disconnected during neurodegenerative disease? Over the past year, we have made three significant advances in our studies of the development of neural connectivity. First, we have used genome-wide expression profiling to identify downstream targets for the transcription factor Lola, an axon guidance regulator that is required for all Notch-dependent axon patterning decisions characterized to date. Second, we have found that the mechanisms and principles we have been studying in axon growth and guidance also control dendrite arborization. Third, we have developed FRET-based biosensors to report the in vivo activity of two major outputs of the Abl signaling pathway, Abl kinase and Rac GTPase. We will briefly describe these, in turn. The gene lola encodes more than 20 isoforms of a zinc finger transcription factor by alternative splicing. These forms heterodimerize to form probably well over a hundred protein species with distinct DNA-binding specificities. Our previous studies showed that lola is required for Notch-dependent axon guidance decisions, including longitudinal axon growth and midline crossing regulation in the CNS and development of the ISNb motonerve in the PNS. We have now performed genome-wide expression profiling of lola mutant embryos using microarrays. Gene ontology analysis verified regulation of a number of Notch pathway genes downstream of lola. It also revealed regulation of a variety of genes associated with axon growth, cell migration, signal transduction and cytoskeletal structure. In addition to genes known to act in Notch-dependent guidance processes, a substantial number of these genes were either anonymous ORFs without demonstrated function in any system, or were genes known to act in other guidance processes but not previously shown to interact with Notch. These experiments therefore significantly expand the window of genes available for analysis in our efforts to understand Notch-dependent neural wiring. Of particular interest was our discovery that a key aspect of lola-dependent axon growth is to suppress expression of the conserved actin nucleation factor, Spire. This observation accords well with evidence we published last year that emphasized the importance of balancing different kinds of actin structures in the growth cone to achieve effective axon growth and faithful axon guidance. We wanted to test whether the mechanisms we have described in axons also apply to dendritic development. Two lines of experiment from the past year support that hypothesis, but with interesting differences. First, we examined the role of Abl signaling components, particularly Rac GTPase and the Rac GEF, Trio, in dendritic arborization. We found that increased or decreased function of these genes alters dendritic arborization in vivo, and that these manipulations interact genetically with mutations of Abl itself, as for axons. The nature of the interaction is evidently different in dendrites, however: mutations that give similar phenotypes and interact synergistically in axons were found to give opposite effects in dendrites and interact antagonistically. The mechanistic basis for this difference is not yet clear. We also examined the role of Lola in dendrites, and its interaction with Spire (in collaboration with D. van Meyel, McGill Univ). Again, we found dendritic phenotypes consistent with what we had demonstrated previously in axons, and as in axons, we found genetic and molecular evidence that a key role of Lola in dendrogenesis is to suppress Spire function (ms submitted). These data support the idea that the molecules and principles governing dendritic arborization are closely akin, but not identical, to those acting in axons. The next phase of our studies will require in vivo monitoring of signal transduction in growth cones to correlate genetic inputs with cytoskeletal dynamics. To this end we have developed FRET-based in vivo biosensors that report the two key outputs of the Abl signaling pathway. One reports Rac activity using a modified form of the Raichu-Rac reporter first developed for mammalian cells. This reporter is identical to one developed and validated independently for flies by D. Montell. Our evidence, in concordance with hers, shows that FRET activity is increased by stimulation of Rac activity and suppressed by expression of a dominant-negative Rac. The other is a reporter for Abl tyrosine kinase activity, and was originally developed for mammalian Abl. We have verified that we can detect the Abl FRET signal in fly cells, that it is stimulated by expression of activated Abl and suppressed in an Abl mutant, and also that it is suppressed by administration of the Abl kinase inhibitor Gleevec. This gives us the reagents we need to detect the crosstalk between Abl and Trio directly, and thereby to validate or falsify the hypothesis that these act as parallel outputs of the Abl signaling network. It also gives us the reagents to correlate Abl kinase activity and Rac GTPase activity with specific aspects of growth cone morphology and motility in vivo, which is a major goal of our overall research program.

我们试图回答两个问题：神经元在发育过程中是如何连接的，以及为什么它们在神经退行性疾病中会断开连接？ 在过去的一年里，我们在神经连接发展的研究中取得了三项重大进展。首先，我们使用全基因组表达谱来识别转录因子Lola的下游靶点，Lola是一种轴突指导调节剂，是迄今为止所有Notch依赖性轴突图案化决定所需的。其次，我们发现我们一直在研究的轴突生长和引导的机制和原则也控制树突树枝化。第三，我们已经开发了基于FRET的生物传感器来报告Abl信号通路的两个主要输出的体内活性，Abl激酶和Rac GT3。我们将依次简要描述这些。 基因lola通过选择性剪接编码超过20种锌指转录因子亚型。这些形式异二聚化形成可能超过一百种具有不同DNA结合特异性的蛋白质种类。我们以前的研究表明，lola是Notch依赖性轴突指导决策所必需的，包括CNS中的纵向轴突生长和中线交叉调节以及PNS中ISNb运动神经的发育。我们现在已经使用微阵列对lola突变胚胎进行了全基因组表达谱分析。基因本体分析验证了lola下游的许多Notch途径基因的调控。它还揭示了与轴突生长，细胞迁移，信号转导和细胞骨架结构相关的各种基因的调节。除了已知在Notch依赖性引导过程中起作用的基因之外，这些基因中的大量基因要么是在任何系统中未证实功能的匿名ORF，要么是已知在其他引导过程中起作用但先前未显示与Notch相互作用的基因。因此，这些实验显着扩大了我们在理解Notch依赖性神经布线的过程中可用于分析的基因窗口。特别感兴趣的是，我们发现，一个关键方面的萝拉依赖性轴突生长是抑制保守的肌动蛋白成核因子，螺旋的表达。这一观察结果与我们去年发表的证据非常一致，这些证据强调了平衡生长锥中不同类型肌动蛋白结构的重要性，以实现有效的轴突生长和忠实的轴突引导。 我们想测试我们在轴突中描述的机制是否也适用于树突发育。过去一年的两项实验支持了这一假设，但存在有趣的差异。首先，我们研究了Abl信号传导组分，特别是Rac GTdR和Rac GEF，Trio在树突状树枝化中的作用。我们发现，增加或减少这些基因的功能改变树突状分支在体内，这些操作与Abl本身的突变，轴突的遗传相互作用。相互作用的性质在树突中明显不同，然而：在轴突中产生相似表型和协同作用的突变被发现在树突中产生相反的作用，并拮抗性地相互作用。这种差异的机制基础尚不清楚。我们还研究了Lola在树突中的作用，以及它与Spire的相互作用（与D。货车Meyel，麦吉尔大学）。再次，我们发现树突表型与我们以前在轴突中证明的一致，并且在轴突中，我们发现遗传和分子证据表明Lola在树突发生中的关键作用是抑制Spire功能。这些数据支持了这样一种观点，即支配树突分支的分子和原理与轴突中的分子和原理非常相似，但不完全相同。 我们研究的下一阶段将需要在体内监测生长锥中的信号转导，以将遗传输入与细胞骨架动力学相关联。为此，我们开发了基于FRET的体内生物传感器，其报告Abl信号通路的两个关键输出。一个报告Rac活性使用的Raichu-Rac报告基因的修改形式，首先开发的哺乳动物细胞。该报告基因与D.蒙特尔我们的证据，在与她的一致，表明FRET活性增加的Rac活性的刺激和抑制的显性负Rac的表达。另一个是一个报告的Abl酪氨酸激酶活性，最初是为哺乳动物的ADAPTIVE。我们已经证实，我们可以检测到的Abl FRET信号在苍蝇细胞中，它是由激活的Abl的表达刺激和抑制在Abl突变体，它也被抑制的Abl激酶抑制剂格列卫的管理。这为我们提供了直接检测Abl和Trio之间的串扰所需的试剂，从而验证或证伪了这些作为Abl信号网络的并行输出的假设。它还为我们提供了将Abl激酶活性和Rac GTdR活性与体内生长锥形态和运动性的特定方面相关联的试剂，这是我们整体研究计划的主要目标。