The formation and maturation of chemical synapses is essential to all major functions of the nervous system. Synaptogenesis represents a complex process of coordinated morphogenesis governed by highly conserved signaling pathways and intracellular effector proteins. Over the past decade, accumulated evidence revealed that this process is under regulatory control of various post-transcriptional mechanisms, from RNA trafficking to local translation, that offer the spatial acuity needed for the complex architectureof neurons and neural circuits. Amongst the sequence-specific regulators that may shape the translation and stability of key neuronal mRNAs, microRNAs (miRs) have recently emerged as a rich potential source. This large and diverse class of non-coding RNA is expressed abundantly in the nervous system, with hundreds of miRs localized to different brain regions, specific neuronal subpopulations and synaptic compartments. Despite impressive advances in the sequencing and informatics technologies required to detect miRs and their targets within the transcriptome, a comprehensive analysis of miR functions required for synaptogenesis was largely limited to in vitro systems where the endogenous cell-cell interaction and developmental signals are lost. In order to address this challenge, we spent the first cycle of funding on this grant developing genetic tools for conditional manipulation and detection of miR function in a model system that has proven to be a powerful model of excitatory glutamatergic synaptic transmission: the Drosophila neuromuscular junction (NMJ). We used our toolkit to dissect a novel trans-synaptic mechanism by which miR-8 regulates coordinated morphogenesis of pre- and post-synaptic compartments during development, in addition to experiments to prove the efficacy of these tools for a variety of experimental questions. We then created a collection of improved reagents to analyze synapse- regulatory function for 141 miRs, and completed a screen revealing that there are dozens of well-conserved miRs required for distinct aspects of NMJ formation. This pioneering effort has put us in a unique position to map out the spatio-temporal landscape of miR regulation for each of the stages of synapse formation and maturation in this system. We have identified two particular miRs that exert potent and reciprocal regulatory effects on opposite sides of the synapse: miR-34 is required in muscle to control addition and spacing of presynaptic boutons, whereas, miR-137 is required in neurons to restrict the overall growth of presynaptic arbors. Both miR-34 and miR-137 show striking conservation to genes implicated in neurological and psychiatric disease, confirming that they are functionally relevant across phylogeny, and suggesting that deeper analysis in our model can be used to ask if the downstream mechanisms are also conserved. In parallel with experiments to define the miRs that act in motor neurons and muscles to shape NMJ development, we will pursue a multi-level analysis of phenotypes and miR target gene networks in order to understand the cellular and molecular logic of the mechanisms by which conserved miRs control synaptogenesis.
Precise regulation of synapse development is essential for normal nervous system function. Although many highly conserved signaling pathways are known to control the cell biological effector genes that construct and maintain synapse structure, we know much less about the regulatory mechanisms that orchestrate deployment of these genes and pathways. We performed a pioneering in vivo screen for microRNAs required for the formation of excitatory glutamatergic synapses in Drosophila; and next, we will define the spatio-temporal specificity of these microRNA functions, and then determine the underlying cellular and molecular mechanisms.
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