Multiplexed Microfluidic Gradients for Axon Guidance

描述(申请人提供)：在神经系统发育过程中，生长轴突对其环境的反应对神经元之间复杂的连接模式的形成至关重要。生长和导向因子结合细胞外基质影响轴突生长的速度和方向。尽管在确定影响轴突生长的因素以及轴突如何单独响应这些因素方面已经取得了很大进展，但对于轴突如何响应多种因素的综合影响还知之甚少。作为目前体内分子成像方法的补充，我们建议开发一个体外环境，潜在地模拟体内发现的一些复杂性，特别是前视觉通路的发展。在这个系统中，轴突轨迹很简单，已经识别了多种相关的引导分子(其中许多是在体外用外植体测试的)，并且一个常见的失明原因(视神经发育不良)与这一过程中的缺陷有关。此外，在视网膜的平面解剖结构上发现的引导分子的图案非常适合通过微图案化和微流体技术进行模拟。这种模仿将通过结合可扩散梯度的微流体图案化和底物结合的轴突寻路线索的激光图案化来完成，包括轴突引导因子和细胞外基质分子。作为高度同质细胞群的来源，我们将分离小鼠视网膜神经节细胞(RGC)，这是一种对Netrin-1梯度反应的细胞类型。对于旨在最大限度地提高细胞完整性的实验(分离程序正在损害细胞)，我们将使用视网膜外植体，并将微流体从轴突的胞体中分离出来。视网膜神经节细胞(或其轴突)将暴露于各种先前已被证明影响其体内轴突生长的可溶性因子中。新的微流控系统将使我们能够测试多种因素对视网膜节细胞轴突生长方向和速度的综合影响。这些实验将使我们能够定量地检查在前视觉通路的发展中支配轴突寻路的基本原理。这些信息将有助于更好地了解轴突发育缺陷的基础，这些缺陷会改变神经系统的组织和功能。 与公共健康相关：轴突引导的研究仅限于单一信号梯度。在活体中，神经元遇到多个信号(结合到底物和溶液中)，必须在信息丰富的环境中做出选择。这项拟议的研究将探索前所未有的复杂性以及前所未有的测量精度的轴突引导相互作用，这将显著加深我们在细胞和分子水平上对轴突生长、神经发育的整体理解，并可能为治疗神经疾病、神经再生和血管病理提供见解。

DESCRIPTION (provided by applicant): During development of the nervous system the response of growing axons to their environment is critical to the formation of the complex wiring pattern between neurons. Growth and guidance factors combined with extracellular matrices influence the speed and direction of axonal growth. Although much progress has been made in identifying the factors that influence axonal growth, as well as how axons respond to these factors individually, much less is known about how axons behave in response to the combined effects of multiple factors. As a complementary approach to present in vivo molecular imaging approaches, we propose to develop an in vitro environment that potentially mimics some of the complexity found in vivo, in particular the development of the anterior visual pathway. In this system, the axon trajectories are simple, multiple relevant guidance molecules have been identified already (many tested with explants in vitro), and a common cause of blindness (Optic Nerve Hypoplasia) is associated with defects in this process. Additionally, the patterns of guidance molecules found on the flat anatomy of the retina are ideally suited to mimicking by micropatterning and microfluidics techniques. This mimicry will be accomplished by combining microfluidics patterning of diffusible gradients and laser patterning of substrate-bound axon pathfinding cues, including axon guidance factors and extracellular matrix molecules. As a source of highly homogeneous cell populations, we will isolate mouse retinal ganglion cells (RGCs), a cell type that responds to Netrin-1 gradients. For experiments designed to maximize the integrity of the cells (isolation procedures are damaging to cells), we will use retinal explants and we will microfluidically isolate the axons from their somas. RGCs (or their axons) will be exposed to various soluble factors that have previously been shown to affect their axon growth in vivo. The new microfluidic systems will allow us to test the combinatorial effects of multiple factors on the direction and speed of axonal growth of RGCs. These experiments will allow us to quantitatively examine the basic principles that govern axon pathfinding in the development of the anterior visual pathway. This information will help to better understand the basis of developmental defects in axon growth that alter the organization and function of the nervous system. PUBLIC HEALTH RELEVANCE: The study of axon guidance has been limited to a single signal gradient. In vivo, neurons encounter multiple signals (both bound to substrate and in solution) and must make choices in an information rich environment. The proposed study will probe axon guidance interactions of unprecedented complexity as well as with unprecedented measurement precision, which will significantly further our understanding of axon growth on a cellular and molecular level, neural development as a whole, and may provide insight on treating neurological disorders, nerve regeneration, and vascular pathologies.