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，迁移到发育中的大脑中所需的位置，轴突和树突的成熟，以及建立适当的突触连接是胚胎神经元发育的基本过程。在这些重叠和协调的发育过程中，神经细胞的发育始于神经元建立轴突和树突身份的过程，这一结构对神经元的输入/输出功能至关重要。在过去的十年中，许多人致力于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一生，是依赖经验的可塑性的基础。此外，影响树突发育和成熟的异常在智力低下和自闭症谱系障碍等严重疾病中起着关键作用。识别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特征的分子机制在很大程度上仍未被探索。目前关于神经元发育的机制范式是，一个轴突的轴突分化伴随着其他轴突的树突形成，这表明树突的发育可能是一种默认的途径。这一建议提出了“默认树突”的另一种假说，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，单个未分化轴突中cAMP活性的局部升高决定了它的轴突命运17、19。其次，来自形成轴突的负信号导致同一细胞3、18-20中的所有其他轴突的轴突形成受到抑制。最后，局部的cGMP特异性信号事件19、25、26决定了这些突起的树突命运。在这个建议中，我们确定了cGMP特异性的树突促进事件。我们提出了三个具体的目标来在体外和体内验证我们的假设。首先，我们直接测定了cGMP在培养的啮齿动物交感神经元44、45中诱导的树突形成，其中树突分化是一个诱导过程。接下来，我们研究cGMP信号的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对发育中的胚胎大脑皮质祖细胞树突发育的促进作用。在这些研究中，我们使用了广泛的方法，结合了使用宫内电穿孔的胚胎遗传操作、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。决定树突状细胞命运的cGMP特异性事件，如本研究中所确定的，直接与自闭症谱系障碍的神经病理学有关。

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.