COMPLEXITY OF IN VIVO AXON GUIDANCE

该项目的长期目标是学习如何在活体内操纵轴突生长。在许多情况下，这将是神经系统损伤后治疗的重要组成部分。此外，移植神经元用于治疗各种中枢神经系统疾病的可能性可能依赖于这些神经元将轴突发送到适当的靶点。发育过程中生长的轴突所遵循的路径受引导机制的调节，这些引导机制由沿路径非均匀分布的分子线索组成。已经提出了吸引和排斥引导线索的梯度的特殊作用。为了实现这一长期目标，有必要详细了解引导线索的梯度如何指导轴突的生长。最近的研究进展表明，3个非扩散梯度分子参与了蟑螂胚胎腿部Til先锋轴突的生长调控，并证实了它们的体内功能。这些分子定位于轴突生长的底物，包括基底膜上的2个分子和表皮上皮细胞表面的L分子。这些指导线索和生长锥体之间相互作用的复杂性比预期的要大。这种复杂性表现在存在多个功能冗余的线索，以及这些线索可能相互作用的事实中。蟑螂胚胎是研究这一发育事件复杂性的理想系统。其解剖结构的简单性使人们能够在活体制剂中观察到腿部已识别的先锋轴突的近端生长。在这项提案中，这些线索中的每一个都将被分子表征，它们在体内和体外指导轴突生长的能力的细节将被检测，以及它们之间的体内相互作用。第四个分子沿轴突生长路径呈梯度分布，已被确定，并建议进行实验以检验其在轴突引导中的作用。像许多发育事件一样，轴突指导也可能在系统发育上是保守的。从对这种昆虫的研究中获得的信息很可能与理解哺乳动物的这些过程非常相关。

The long-term goal of this project is to learn how to manipulate axon growth in vivo. There are many instances when this would be an essential part of a therapeutic treatment after injury to the nervous system. In addition, the possible use of transplanted neurons to treat various CNS diseases may depend on these neurons sending axons to appropriate targets. The path growing axons follow during development is regulated by guidance mechanisms consisting of molecular cues that are non- uniformly distributed along the path. A particular role for gradients of attracting and repelling guidance cues has been proposed. A detailed understanding of how gradients of guidance cues direct the growth of axons is necessary to achieve this long-term goal. Recent progress has resulted in the identification and demonstration of in vivo function of 3 non-diffusible gradient molecules involved in the guidance of growth of the Til pioneer axons in the legs of the cockroach embryo. These molecules, localized to the substrate over which the axons grow, include 2 in the basement membrane and l on the surface of the epidermal epithelial cells. The complexity of the interactions between these guidance cues and the growth cone is greater than had been expected. This complexity is expressed in the fact that multiple and functionally redundant cues exist and in that these cues may interact with one another. The cockroach embryo is an ideal system in which to study the complexity of this developmental event. Its anatomical simplicity enables the proximal growth of identified pioneer axons in the leg to be observed in living preparations. In this proposal each of these cues will be molecularly characterized, the details of their ability to guide axon growth both in vivo and in vitro will be examined, as will be the in vivo interactions among them. A fourth molecule distributed in a gradient along the path of axon growth has been identified and experiments are proposed to examine its role in axon guidance. Like many developmental events, axon guidance is also likely to be phylogenetically conserved. The information obtained from studies of this insect are likely to be very relevant to understanding these processes in mammals.