Netrin5 in Mammalian Neurodevelopment

描述（由申请人提供）：本提案的目标是确定一个新的Netrin基因家族成员Netrin5在哺乳动物神经系统运动和感觉神经元发育中的功能。脊髓和脑神经中运动神经元和感觉神经元的发育得到了很好的研究；然而，主要的问题仍然存在，包括运动轴突如何识别腹侧运动出口点，感觉轴突如何识别背根进入区，以及中枢和周围神经系统之间的边界如何建立。这一边界的性质尚不清楚。没有观察到物理屏障，如基底膜，并且这些重要功能背后的信号定义不清。我们最近发现了Netrin基因家族的一个新成员，它可能就是这样一个信号。众所周知，在发育中的神经系统中，Netrin具有指导轴突引导和细胞迁移的作用，我们在研究运动轴突引导中的Netrin信号传导过程中，在小鼠中发现了Netrin5。Netrin5基因在人类基因组和小鼠中保守，在小鼠胚胎脊髓的运动出口点和背根进入区发现的细胞群中表达，并与感觉三叉神经和可能的其他颅神经有关。这些细胞似乎是边界帽细胞，一种短暂的神经嵴来源的细胞群，其功能是将运动神经元细胞体限制在腹角，并促进背根雪旺细胞和背根神经节伤害性神经元群体。Netrin5的表达模式表明这种发育信号在运动和感觉神经元的发育以及外周轴突进出脊髓的引导中起作用，并且可能在脑神经中具有类似的功能。我们已经构建了用于体外研究的表达结构，以及用于体内评估其功能的缺乏Netrin5表达的敲除小鼠。在我们的第一个目标中，我们将检查缺乏Netrin5的运动和感觉神经元的发育。这些研究将根据边界帽细胞的已知功能，如将运动神经元胞体限制在腹侧角或分化为背根神经节中发现的伤害性神经元。我们还将研究已知的Netrin依赖过程，其中特定的Netrin基因尚未确定，例如滑车神经引导。除了解剖研究外，我们还将评估小鼠的运动和感觉行为，以确定Netrin5缺失的功能后果。在我们的第二个目标中，我们将在体外确定NETRIN5蛋白对运动和感觉轴突生长和引导的影响，并确定所需的Netrin受体。这些研究将由netrin受体的表达模式提供信息，体外结果将通过从netrin受体敲除小鼠中培养的神经元以及对相关受体敲除菌株可能的表型进行体内评估来证实。总之，这些实验将确定Netrin5在哺乳动物神经发育中的作用，并解决有关运动和感觉神经元发育的重要问题。

DESCRIPTION (provided by applicant): The goal of this proposal is to determine the function of a new Netrin gene family member, Netrin5, in the development of motor and sensory neurons in the mammalian nervous system. The development of motor and sensory neurons in the spinal cord and cranial nerves is well studied; however, major questions remain, including how motor axons identify the ventral motor exit point, how sensory axons identify the dorsal root entry zone, and how the boundary between the central and peripheral nervous system is established. The nature of this boundary is unclear. No physical barrier, such as a basal lamina, has been observed, and the signals that underlie these important functions are poorly defined. We recently identified a new member of the Netrin family of genes that may be such a signal. Netrins are well known to direct axon guidance and cell migration in the developing nervous system, and we identified Netrin5 in mice in the course of our studies on Netrin signaling in motor axon guidance. The gene is conserved in the human genome and in mice, Netrin5 is expressed in a population of cells found at the motor exit points and dorsal root entry zones in the mouse embryonic spinal cord, and is associated with the sensory trigeminal nerve and possibly other cranial nerves. These cells appear to be boundary cap cells, a transient neural crest-derived cell population that functions to restrict motor neuron cell bodies to the ventral horn and to contribute to dorsal root Schwann cell and dorsal root ganglia nociceptive neuron populations. The expression pattern of Netrin5 suggests a role for this developmental signal in motor and sensory neuron development and peripheral axon guidance into and out of the spinal cord, and possibly similar functions in cranial nerves. We have generated expression constructs for in vitro studies, and knockout mice lacking Netrin5 expression for an in vivo assessment of its function. In our first Aim, we will examine motor and sensory neuron development in the absence of Netrin5. These studies will be informed by the known function of boundary cap cells, such as restricting motor neuron cell bodies to the ventral horn or differentiating into nociceptiv neurons found in the dorsal root ganglia. We will also examine known Netrin-dependent processes for which the specific Netrin gene responsible has not been determined, such as trochlear nerve guidance. In addition to anatomical studies, we will assess motor and sensory behaviors in the mice to determine the functional consequences of the loss of Netrin5. In our second Aim, we will determine the effect of NETRIN5 protein on motor and sensory axon outgrowth and guidance in vitro, and determine the Netrin receptors required. These studies will be informed by the expression patterns of netrin receptors, and in vitro results will be confirmed genetically with neurons cultured from Netrin receptor knockout mice and by an in vivo assessment of possible phenocopy in the relevant receptor knockout strains. Together, these experiments will determine the role of Netrin5 in mammalian neurodevelopment and address important, unanswered questions about motor and sensory neuron development.