Mechanisms of axon guidance during development

目前，我们对轴突生长和指导的大部分努力都集中在理解信号模块的作用，这些模块将生长锥表面的受体信号转化为轴突尖端的指导运动。我们特别关注Abl酪氨酸激酶信号传导途径，其在许多最常见的、遗传上保守的指导受体的下游起着核心作用。在一个完全出乎意料的转折中，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器官来执行的。因此，在目标1下，我们有以下发现： 1. 关键的Abl效应子，肌动蛋白调节蛋白Enabled，选择性地与发育中的果蝇感光神经元中的顺式高尔基体隔室相关。我们已经通过标准荧光显微镜、超分辨率荧光和免疫电子显微镜显示了这一点。 2. Abl激酶的功能丧失或其拮抗剂Enabled的功能获得，导致高尔基体的碎片化和高尔基池在感光神经元的细胞体内的重新定位。活体成像表明，降低Abl活性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肌动蛋白细胞骨架的β-依赖性调节介导的。 4. 两条证据表明，Abl途径突变对轴突导向的影响部分是由其对分泌的影响介导的。首先，直接干扰高尔基体功能的突变产生模仿Abl突变体的轴突缺陷。第二，基因测试表明，真正的分泌突变体的行为下游的Abl激酶的途径，导致轴突图案。 总之，这些数据有力地表明，轴突表型的Abl突变体是由于，在一定程度上，通过调节肌动蛋白的结构和动力学的β-依赖性调节的分泌装置的Abl，工作。 在脊椎动物和无脊椎动物中，Abl是轴突图案化、细胞极性和上皮组织和完整性的中心调节剂。它是两种常见的人类白血病的致癌基因，最近的证据表明它在帕金森病的病因学中起着关键作用。三十年来，Abl的研究集中在其对皮质肌动蛋白细胞骨架的影响。我们的研究结果表明，现在必须重新考虑大量关于Abl的工作，以确定其影响中有多少实际上是部分或全部通过其对蛋白质分选，运输和分泌的影响来介导的。 在过去的一年里，我们还根据目标2启动了实验，开发了一个系统，用于单个TSM神经元的实时成像，因为它们是发育中的苍蝇翅膀的L1神经的先驱。预蛹翅盘从苍蝇表达荧光标记的肌动蛋白细胞骨架或质膜在TSM的旋转盘共聚焦显微镜检查和可视化。初步观察表明，原位先驱神经元的形态与之前对固定样本的分析或对培养物中生长的轴突成像得出的结果有根本不同。事实上，只有当轴突不延伸时，才能观察到通常与生长锥相关的大而平的层状形态;这是与静止生长锥在其轨迹中的选择点相关的形态。此外，与普遍认为板状伪足提供生长动力的观点相反，我们发现快速延伸的轴突几乎没有板状伪足。相反，轴突的延伸是由丝状伪足的选择性保留引起的。最后，初步的数据表明，稳定和选择性保留的主导丝状伪足是由它们的相关性与当地分布的指导线索三角洲的底层翅上皮。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。

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.