Mechanisms of axon guidance during development

发育过程中轴突引导的机制

• 批准号: 8940066
• 负责人: edward giniger
• 金额: $ 83.48万
• 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/8940066
• 关键词: Actins; Animals; Axon; Biological Assay; Cell Polarity; Cell membrane; Cell physiology; Confocal Microscopy; Cues; Cytoskeleton; Data; Defect; Development; Drosophila genus; Epithelial; Epithelium; Etiology; Filopodia; Fluorescence; Fluorescence Microscopy; Frequencies; Genetic; Genetic screening method; Golgi Apparatus; Growth; Growth Cones; Human; Image; Immunoelectron Microscopy; In Situ; Individual; Invertebrates; Life; Mediating; Metric; Modeling; Molecular; Morphogenesis; Morphology; Mutation; Nerve; Neurodegenerative Disorders; Neurons; Oncogenes; Organism; Output; Parkinson Disease; Pathway interactions; Pattern; Phenotype; Phosphotransferases; Photoreceptors; Play; Protein Tyrosine Kinase; Proteins; Receptor Signaling; Regulation; Resolution; Role; Sampling; Signal Pathway; Signal Transduction; Sorting - Cell Movement; Speed; Structure; Surface; Translating; Vertebrates; Wing; Work; axon growth; axon guidance; cell motility; fly; gain of function; genetic regulatory protein; leukemia; loss of function; mutant; neuronal cell body; notch protein; receptor; research study; trafficking

Most of our effort on axon growth and guidance currently focuses on understanding the action of the signaling modules that translate receptor signals at the growth cone surface into guided motility of the axon tip. We focus in particular on the Abl tyrosine kinase signaling pathway, which plays a central role downstream of many of the most common, phylogenetically-conserved guidance receptors. In a completely unexpected twist, in the past year we discovered that the guidance function of Abl is executed, in part, through regulation of the secretory apparatus. Thus, under Aim 1 we have made the following discoveries: 1. The key Abl effector, the actin-regulatory protein Enabled, is selectively associated with the cis-Golgi compartment in developing Drosophila photoreceptor neurons. We have shown this by standard fluorescence microscopy, super-resolution fluorescence and by immunoelectron microscopy. 2. Loss-of function of Abl kinase, or gain-of-function of its antagonist Enabled, cause fragmentation of the Golgi apparatus, and relocalization of Golgi cisternae within the cell bodies of photoreceptor neurons. Live imaging demonstrates that reducing Abl activity increases the frequency of splitting of Golgi cisternae, and decreases the frequency of fusions. 3. Genetic and pharmacological experiments demonstrate that the effect of Abl on the Golgi structure and distribution is mediated through Abl-dependent regulation of the actin cytoskeleton. 4. Two lines of evidence suggest that the effect of Abl pathway mutations on axon guidance are mediated, in part, by their effects on secretion. First, mutations that interfere directly with Golgi function produce axonal defects that mimic those of Abl mutants. Second, genetic tests show that bona fide secretory mutants act downstream of Abl kinase in the pathway leading to axon patterning. Together, these data strongly suggest that the axonal phenotypes of Abl mutants are due, in part, to modulation of the secretory apparatus by Abl, working through the Abl-dependent regulation of actin structure and dynamics. Abl is a central regulator of axon patterning, cell polarity and epithelial organization and integrity in both vertebrates and invertebrates. It is the causative oncogene for two common forms of human leukemia, and recent evidence suggest it has a critical role in the etiology of Parkinson's disease. For three decades, studies of Abl have focused on its effects on the cortical actin cytoskeleton. Our results suggest that a vast body of work on Abl must now be reconsidered to ascertain how many of its effects are actually mediated, in part or in whole, through its effects on protein sorting, trafficking and secretion. In the past year, we have also initiated experiments under Aim 2 by developing a system for live imaging of single TSM neurons as they pioneer the L1 nerve of the developing fly wing. Prepupal wing discs from flies expressing fluorescent markers of the actin cytoskeleton or the plasma membrane in TSM are explanted and visualized by spinning disc confocal microscopy. Initial observations reveal that the morphology of pioneer neurons in situ is fundamentally different from the picture that has been derived from previous analyses of fixed samples, or from imaging axons growing in culture. The large, flat, lamellar morphology typically associated with growth cones is in fact observed only when the axon is not extending; it is the morphology associated with stationary growth cones at a choice point in their trajectory. Moreover, contrary to the common view that lamellipodia provide the motive force for growth we find that rapidly extending axons have few if any lamellipodia. Rather, axon extension arises from selective retention of filopodia. Finally, preliminary data suggest that stabilization and selective retention of leading filopodia is determined by their correlation with the local distribution of the guidance cue Delta on the underlying wing epithelium. We are currently developing metrics for quantifying cytoskeletal structures in these growth cones and analyzing their correlation with growth cone speed and direction.

我们目前在轴突生长和引导方面的大部分工作集中在了解信号模块的作用，这些信号模块将生长锥体表面的受体信号转化为轴突尖端的引导运动。我们特别关注Abl酪氨酸激酶信号通路，它在许多最常见的、系统发育保守的指导受体下游发挥着核心作用。完全意想不到的是，在过去的一年里，我们发现Abl的指导功能部分是通过调节分泌装置来执行的。因此，在目标1下，我们取得了以下发现： 1.在发育中的果蝇光感受器神经元中，关键的abl效应器，即肌动蛋白调节蛋白，选择性地与顺式高尔基体相关联。我们已经用标准荧光显微镜、超分辨荧光和免疫电子显微镜证明了这一点。 2.Abl激酶功能丧失或其拮抗剂功能增强，导致高尔基体碎裂，高尔基池在光感受器神经元胞体内重新定位。活体成像显示，降低Abl活性会增加高尔基体池分裂的频率，并降低融合的频率。 3.遗传学和药理学实验表明，Abl对高尔基体结构和分布的影响是通过Abl依赖的肌动蛋白细胞骨架的调节而实现的。 4.两条证据表明，Abl通路突变对轴突引导的影响部分是通过它们对分泌的影响来介导的。首先，直接干扰高尔基体功能的突变会产生类似Abl突变体的轴突缺陷。其次，基因测试表明，真正的分泌型突变体在导致轴突形成的途径中作用于Abl激酶的下游。 综上所述，这些数据强烈表明，Abl突变体的轴突表型在一定程度上是由于Abl调节分泌装置，通过Abl依赖的肌动蛋白结构和动力学调节发挥作用。 ABL是脊椎动物和无脊椎动物轴突形态、细胞极性、上皮组织和完整性的中央调节因子。它是两种常见人类白血病的致癌基因，最近的证据表明，它在帕金森氏病的病因学中起着关键作用。三十年来，ABL的研究一直集中在其对皮质肌动蛋白细胞骨架的影响上。我们的结果表明，现在必须重新考虑大量关于Abl的工作，以确定其有多少影响实际上是通过其对蛋白质分类、运输和分泌的影响而部分或全部介导的。 在过去的一年里，我们还在目标2下启动了实验，开发了一个系统，用于对单个TSM神经元进行实时成像，因为它们是发育中的苍蝇翅膀的L1神经的先驱。用旋转圆盘共聚焦显微镜观察表达TSM中肌动蛋白细胞骨架或质膜的荧光标记物的苍蝇的预蛹翼盘。初步观察显示，原位先锋神经元的形态与先前对固定样本的分析或培养中生长的轴突成像所得出的图像有根本不同。事实上，只有当轴突没有伸展时，才能观察到与生长锥有关的大的、扁平的、片层的形态；这是与静止的生长锥在其轨迹上的一个选择点有关的形态。此外，与普遍认为片状脂肪提供生长动力的观点相反，我们发现快速伸展的轴突几乎没有片状脂肪。相反，轴突的延伸是由于选择性地保留了丝状足突。最后，初步数据表明，前导丝状足细胞的稳定和选择性保留取决于它们与引导线索Delta在下层翼上皮上的局部分布的相关性。我们目前正在开发量化这些生长锥中的细胞骨架结构的指标，并分析它们与生长锥速度和方向的相关性。