Molecular Mechanisms of Dendrite Formation During Embryonic Neuronal Development

描述（申请人提供）：轴突和树突的形成和定向生长，向发育中的大脑中所需位置的迁移，轴突和树突乔木的成熟，以及适当突触连接的建立是胚胎神经元发育的基本过程。由于这些重叠且协调良好的发育过程，神经元细胞的发育始于神经元建立轴突和树突身份的过程，这是神经元输入/输出功能的关键结构。在过去的十年中，人们已经努力阐明控制轴突起始和生长的分子和细胞机制1-23。树突的形态和功能分化贯穿整个生命，是经验依赖可塑性的基础。此外，影响树突发育和成熟的异常在智力迟钝和自闭症谱系障碍等严重疾病中起着关键作用。确定决定树突发育早期事件的信号通路对于将这些机制转化为临床应用具有重要意义。尽管如此，在胚胎发育过程中建立树突同一性的分子机制在很大程度上仍未被探索。目前，神经元发育的机制范式是，一个神经突的轴突分化伴随着其他神经突的树突形成，这表明树突发育可能代表了一种默认途径。该建议提供了“默认树突”的另一种假设，并提出了轴突/树突建立的“三步”模型，该模型由第二信使cAMP和cGMP活性的相反变化驱动。首先，单个未分化神经突中camp活性的局部升高决定了其轴突的命运17,19。其次，从形成轴突传播的负信号导致同一细胞中所有其他神经突的轴突形成受到抑制3,18-20。最后，局部cgmp特异性信号事件19,25,26决定了这些神经突的树突命运。在这个建议中，我们确定cgmp特异性树突促进事件。我们提出了三个具体的目标来验证我们的假设在体外和体内。首先，我们在培养的啮齿动物交感神经元中直接确定cgmp诱导的树突形成，其中树突分化是一个可诱导的过程。接下来，我们研究cGMP信号下游和上游可能的树突特异性决定因素。最后，我们在体内研究了cgmp对发育中的胚胎脑皮层祖细胞树突发育的促进作用。在这些研究中，我们采用了广泛的方法，结合胚胎基因操作，使用子宫内电穿孔，小鼠遗传学，生物化学，材料工程，延时显微镜和荧光共振能量转移（FRET）成像。cGMP特异性事件决定树突的命运，正如本研究所确定的那样，与自闭症谱系障碍的神经病理学直接相关。

DESCRIPTION (provided by applicant): The formation and directed growth of axon and dendrite, migration to a desired location in the developing brain, maturation of axonal and dendritic arbors, and establishment of proper synaptic connections are essential processes during embryonic neuronal development. With these overlapping and well coordinated developmental processes, the development of a neuronal cell begins with a process in which the neuron establishes axon and dendrite identities, an architecture that is critical for the input/output functions of the neuron. In the last decade, much effort has been made towards the elucidation of the molecular and cellular mechanisms that control axon initiation and outgrowth1-23. The morphological and functional differentiation of dendrites persists throughout life and underlies experience-dependent plasticity. Moreover, aberrations affecting dendrite development and maturation play key role in severe disorders such as mental retardation and autism spectrum disorders. The identification of the signaling pathways that determine the early events in dendrite development is of great importance for efforts to translate these mechanisms into clinical application. Despite this, the molecular mechanisms that establish the dendritic identity during embryonic development remain largely unexplored. The current mechanistic paradigm for neuronal development is that axon differentiation of one neurite is accompanied by dendrite formation in other neurites, suggesting that dendrite development may represent a default pathway. This proposal offers an alternative hypothesis to "dendrites by default" and proposes a 'three-step' model of axon/dendrite establishment that is driven by opposite changes in the activity of the second messengers cAMP and cGMP. First, local elevation of cAMP-activity in a single undifferentiated neurite determines its axon fate17,19. Second, a negative signal propagating from the forming axon results in inhibition of axon formation in all other neurites of the same cell3,18-20. Last, localized cGMP-specific signaling events19,25,26 determine the dendrite fate of these neurites. In this proposal we identify cGMP-specific dendrite promoting events. We propose three specific aims to test our hypothesis both in vitro and in vivo. First, we directly determine cGMP-induced dendrite formation in cultured rodent sympathetic neurons44,45 in which dendrite differentiation is an inducible process. Next, we examine possible dendrite-specific determinants downstream and upstream of cGMP signaling. Last, we examine cGMP-dependent promotion of dendrite development in cortical progenitors in the developing embryonic brain in vivo. In these studies we employ a wide range of approaches that combine embryonic genetic manipulation using in utero electroporation, mouse genetics, biochemistry, material engineering, time-lapse microscopy and fluorescence resonance energy transfer (FRET) imaging. The cGMP- specific events that determine the dendritic fate, as identified in this study, are directly implicated in the neuropathology of autism spectrum disorders.