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，为什么在神经退行性疾病期间它们会断开连接？ 在过去的一年里，我们在神经连通性发展的研究中取得了三个重大进展。首先，我们使用全基因组表达谱来确定转录因子LOLA的下游靶标，LOLA是迄今为止所有Notch依赖的轴突模式决策所必需的轴突引导调节因子。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理也控制着树突的树枝形成。第三，我们开发了基于FRET的生物传感器来报告Abl信号通路的两个主要输出：Abl激酶和Rac GTP酶在体内的活性。我们将依次对它们进行简要描述。 通过选择性剪接，Lola基因编码了20多种锌指转录因子的亚型。这些形式的异二聚体可能形成100多种蛋白质物种，具有不同的DNA结合特性。我们以前的研究表明，LOLA在Notch依赖的轴突导向决策中是必需的，包括中枢神经系统的纵向轴突生长和中线交叉调节，以及三叉神经节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。基因本体论分析证实了LOLA下游的一些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控。除了已知在Notch依赖的引导过程中起作用的基因外，这些基因中有相当一部分要么是匿名ORF，在任何系统中都没有被证实的功能，要么是已知在其他引导过程中起作用但以前没有显示与Notch相互作用的基因。因此，这些实验极大地扩大了可用于分析的基因窗口，以便我们努力理解Notch依赖的神经连接。特别有趣的是，我们发现依赖LOLA的轴突生长的一个关键方面是抑制保守的肌动蛋白核因子Spire的表达。这一观察结果与我们去年发表的证据非常一致，该证据强调了平衡生长锥体中不同类型的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。过去一年的两条实验路线支持这一假设，但存在有趣的差异。首先，我们研究了Abl信号元件，特别是Rac GTPase和Rac GTP，Trio，在树突分枝中的作用。我们发现，这些基因功能的增加或减少会改变体内树突的树枝形成，并且这些操作与Abl本身的突变存在遗传上的相互作用，就像轴突一样。然而，在树突中，这种相互作用的性质明显不同：提供相似表型并在轴突中协同作用的突变被发现在树突中产生相反的效果，并相互拮抗。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McGill大学的D.van Meyel合作)。再次，我们发现树突状表型与我们之前在轴突中展示的一致，并且像在轴突中一样，我们发现了遗传和分子证据，表明LOLA在树突状细胞发生中的关键作用是抑制螺旋功能(MS提交)。这些数据支持这样一种观点，即支配树突树枝形成的分子和原理与作用于轴突的分子和原理非常相似，但并不完全相同。 我们下一阶段的研究将需要对生长锥中的信号转导进行活体监测，以将遗传输入与细胞骨架动力学联系起来。为此，我们开发了基于FRET的体内生物传感器，报告Abl信号通路的两个关键输出。其中一篇报道了使用一种最初为哺乳动物细胞开发的改良形式的Raichu-RAC报告程序的RAC活性。这份报告与D.Montell为苍蝇独立开发和验证的报告完全相同。我们的证据与她的一致，表明FRET活性通过RAC活性的刺激而增加，而通过显性负性RAC的表达而被抑制。另一种是Abl酪氨酸激酶活性的报告，最初是为哺乳动物Abl开发的。我们已经证实，我们可以在苍蝇细胞中检测到Abl FRET信号，它被激活的Abl表达刺激，而在Abl突变体中被抑制，并且它还被Abl激酶抑制剂格列卫抑制。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 GTP酶活性与体内生长锥形态和运动性的特定方面联系起来的试剂，这是我们整个研究计划的主要目标。