Multiplexed Microfluidic Gradients for Axon Guidance

描述（由申请人提供）：在神经系统发育期间，生长轴突对其环境的反应对于神经元之间复杂布线模式的形成至关重要。 生长和导向因子与细胞外基质结合影响轴突生长的速度和方向。 虽然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取得了很大进展，但对轴突如何响应多个因素的组合效应知之甚少。 作为一种补充的方法，目前在体内的分子成像方法，我们建议开发一种体外环境，有可能模仿一些在体内发现的复杂性，特别是前视觉通路的发展。 在这个系统中，轴突轨迹很简单，已经鉴定出多种相关的引导分子（许多在体外用外植体进行了测试），失明的常见原因（视神经发育不全）与这个过程中的缺陷有关。 此外，在视网膜的平坦解剖结构上发现的引导分子的图案理想地适合于通过微图案化和微流体技术来模仿。 这种模仿将通过结合扩散梯度的微流体图案化和基底结合轴突寻路线索（包括轴突引导因子和细胞外基质分子）的激光图案化来实现。 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梯度有反应的细胞类型。 对于旨在最大限度地提高细胞完整性的实验（分离程序会损害细胞），我们将使用视网膜外植体，并将轴突与其胞体微流体分离。 RGCs（或其轴突）将暴露于各种可溶性因子，这些因子先前已被证明会影响其轴突在体内的生长。 新的微流体系统将使我们能够测试多种因素对RGC轴突生长方向和速度的组合影响。 这些实验将使我们能够定量研究的基本原则，支配轴突寻路的发展中的前视觉通路。 这些信息将有助于更好地理解轴突生长发育缺陷的基础，这些缺陷改变了神经系统的组织和功能。

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.