Glial cells function in diverse aspects of neuronal development and plasticity. Gap junctions also play multiple roles in the nervous system. However the functions of neuron-glia gap junctions in neuronal polarity have not been explored. Here we showed that gap junctions between C. elegans GLR glial cells and RME neurons were required for establishment of RME neuronal polarity. In a genetic screen for RME neuronal circuit formation, we found that loss-of-function in a gap junction regulator, unc-1, induce strong polarity defects. Gap junctions in C. elegans are composed by innexins, two of which unc-7 and unc-9 are expressed in RME neurons. We found that unc-7 unc-9 double mutants displayed similar phenotype as unc-1, suggesting that gap junctions were required for the establishment of RME neuronal polarity. Furthermore, our rescue experiments showed that unc-1 and unc-7 were required in both RME neurons and GLR glial cells, indicating the RME-GLR gap junctions were important for RME polarity. Since microtubule polarity plays a fundamental role in neuronal polarity, we further examined the role of RME-GLR gap junctions in regulation of microtubules using EBP-2::GFP reporter. We found that microtubules orientations were almost evenly mixed in RME dendrites in control animals, but the majority of microtubules were plus-end out cell body in RME dendrites in gap junction mutants. Consistent with this finding, loss-of-function in the minus-end to plus-end motor protein unc-104 was able to suppress the polarity defects in gap junction mutants. In C. elegans, several signal pathways have been reported to regulate microtubule polarity, including the P35-CDK-5 pathway. We found that loss-of-function in each members of the P35-CDK-5 pathway induced similar polarity defects in RME neurons. Double mutants of cdk-5 and unc-7 neither enhanced nor suppressed single mutant phenotypes, suggesting the CDK-5 pathway might function in the gap junction pathway. Previous study showed that the cleavage of P35 was essential for the activation of the P35-CDK-5 pathway. If gap junctions function through the P35-CDK-5 pathway, disruption of gap junctions should affect the cleavage of P35. Indeed, we found the cleavage of P35 was nearly undetectable in gap junction mutants. Furthermore, overexpression of the active version of P35, P25, was able to suppress unc-1 phenotypes. These data suggested that RME-GLR gap junctions functioned through P35-CDK-5 pathway to regulate microtubule polarity. In summary, our study described the role of neuron-glia gap junctions in vivo, and provided a novel mechanism for neuronal polarity, that glial cells directly form gap junctions with neurons to regulate neuronal intracellular pathways.
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