Molecular Mechanisms of Dendrite Formation During Embryonic Neuronal Development

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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.