Poulain Fabienne E
Department of Biological Sciences, University of South Carolina, Coker Life Science Building, 715 Sumter street, Columbia, SC, 29208, USA,
Methods Mol Biol. 2015;1229:469-82. doi: 10.1007/978-1-4939-1714-3_36.
One of the most fascinating questions in the field of neurobiology is to understand how neuronal connections are properly formed. During development, neurons extend axons that are guided along defined paths by attractive and repulsive cues to reach their brain target. Most of these guidance factors are regulated by heparan sulfate proteoglycans (HSPGs), a family of cell-surface and extracellular core proteins with attached heparan sulfate (HS) glycosaminoglycans. The unique diversity and structural complexity of HS sugar chains, as well as the variety of core proteins, have been proposed to generate a complex "sugar code" essential for brain wiring. While the functions of HSPGs have been well characterized in C. elegans or Drosophila, relatively little is known about their roles in nervous system development in vertebrates. In this chapter, we describe the advantages and the different methods available to study the roles of HSPGs in axon guidance directly in vivo in zebrafish. We provide protocols for visualizing axons in vivo, including precise dye labeling and time-lapse imaging, and for disturbing the functions of HS-modifying enzymes and core proteins, including morpholino, DNA, or RNA injections.
神经生物学领域最引人入胜的问题之一是了解神经元连接是如何正确形成的。在发育过程中，神经元伸出轴突，这些轴突在吸引和排斥线索的引导下沿着特定路径延伸，以到达其大脑靶点。这些引导因子大多受硫酸乙酰肝素蛋白聚糖（HSPGs）调控，硫酸乙酰肝素蛋白聚糖是一类细胞表面和细胞外核心蛋白，其上连接有硫酸乙酰肝素（HS）糖胺聚糖。有人提出，HS糖链独特的多样性和结构复杂性以及核心蛋白的多样性会产生一种对大脑布线至关重要的复杂“糖代码”。虽然HSPGs的功能在秀丽隐杆线虫或果蝇中已得到充分表征，但它们在脊椎动物神经系统发育中的作用却知之甚少。在本章中，我们描述了直接在斑马鱼体内研究HSPGs在轴突导向中作用的优势和可用的不同方法。我们提供了在体内可视化轴突的方案，包括精确的染料标记和延时成像，以及干扰HS修饰酶和核心蛋白功能的方案，包括吗啉代寡核苷酸、DNA或RNA注射。
