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.

我们在轴突生长和指导方面的大部分努力目前都集中在理解将生长锥表面的受体信号转化为轴突尖端的引导运动性的信号模块的作用。我们特别关注ABL酪氨酸激酶信号通路，该途径在许多最常见的系统发育保守的引导受体的下游起着核心作用。在一个完全意外的转折中，在过去的一年中，我们发现ABL的指导函数部分是通过对分泌装置的调节执行的。因此，在AIM 1下，我们进行了以下发现： 1。启用肌动蛋白调节蛋白的关键ABL效应子在发育的果蝇光感受器神经元中选择性地与顺式 - 高尔基室有选择性相关。我们通过标准荧光显微镜，超分辨率荧光和免疫电子显微镜证明了这一点。 2。ABL激酶的功能丧失或支持其对拮抗剂的功能获得的功能，导致高尔基体的分裂，并在光感受器神经元细胞体内重新定位高尔基硫酸氢菌。实时成像表明，降低ABL活性会增加高尔基蓄积的分裂频率，并降低融合频率。 3。遗传和药理学实验表明，ABL对高尔基体结构和分布的影响是通过肌动蛋白细胞骨架的ABL依赖性调节介导的。 4。两条证据表明，ABL途径突变对轴突指导的影响部分是由于它们对分泌的影响。首先，直接干扰高尔基体功能的突变会产生模仿ABL突变体的突变。其次，遗传测试表明，善良的分泌突变体在途径中的ABL激酶下游作用，导致轴突构图。 总之，这些数据强烈表明，ABL突变体的轴突表型部分归因于ABL通过ABL依赖性肌动蛋白结构和动力学调节的调节。 ABL是脊椎动物和无脊椎动物中轴突图案，细胞极性和上皮组织以及完整性的中央调节剂。这是两种常见的人白血病形式的病因，最近的证据表明，它在帕金森氏病的病因中具有关键作用。三十年来，对ABL的研究一直集中在其对皮质肌动蛋白细胞骨架的影响。我们的结果表明，现在必须重新考虑大量关于ABL的工作，以确定其实际上或从部分地介导了其对蛋白质分类，贩运和分泌的影响。 在过去的一年中，我们还通过为单个TSM神经元的实时成像开发了AIM 2的实验，因为它们是开发发展蝇翼的L1神经的。来自表达肌动蛋白细胞骨架的荧光标记或TSM中质膜的蝇的前翼圆盘被旋转并通过旋转盘共焦显微镜进行了可视化和可视化。最初的观察结果表明，先锋神经元的形态与先前对固定样品的分析或培养物中生长的成像轴突的图像有根本不同。实际上，仅在轴突不延伸时才观察到大型，平坦的层状形态实际上才观察到。它是与固定生长锥相关的形态，其轨迹的选择点。此外，与普通观点相反，薄片为生长提供了动力，我们发现快速延伸轴突几乎没有薄片。相反，轴突延伸是源自丝状的选择性保留。最后，初步数据表明，稳定和选择性保留领先的丝状病毒取决于它们与基础机翼上皮上指导提示三角洲的局部分布的相关性。我们目前正在开发用于量化这些生长锥中细胞骨架结构的指标，并分析其与生长锥速度和方向的相关性。