Murine glial protrusion transcripts predict localized Drosophila glial mRNAs involved in plasticity.

The polarization of cells often involves the transport of specific mRNAs and their localized translation in distal projections. Neurons and glia are both known to contain long cytoplasmic processes, while localized transcripts have only been studied extensively in neurons, not glia, especially in intact nervous systems. Here, we predict 1,740 localized Drosophila glial transcripts by extrapolating from our meta-analysis of seven existing studies characterizing the localized transcriptomes and translatomes of synaptically associated mammalian glia. We demonstrate that the localization of mRNAs in mammalian glial projections strongly predicts the localization of their high-confidence Drosophila homologs in larval motor neuron-associated glial projections and are highly statistically enriched for genes associated with neurological diseases. We further show that some of these localized glial transcripts are specifically required in glia for structural plasticity at the nearby neuromuscular junction synapses. We conclude that peripheral glial mRNA localization is a common and conserved phenomenon and propose that it is likely to be functionally important in disease.

Systematic analysis of YFP traps reveals common mRNA/protein discordance in neural tissues.

While post-transcriptional control is thought to be required at the periphery of neurons and glia, its extent is unclear. Here, we investigate systematically the spatial distribution and expression of mRNA at single molecule sensitivity and their corresponding proteins of 200 YFP trap lines across the intact Drosophila nervous system. 97.5% of the genes studied showed discordance between the distribution of mRNA and the proteins they encode in at least one region of the nervous system. These data suggest that post-transcriptional regulation is very common, helping to explain the complexity of the nervous system. We also discovered that 68.5% of these genes have transcripts present at the periphery of neurons, with 9.5% at the glial periphery. Peripheral transcripts include many potential new regulators of neurons, glia, and their interactions. Our approach is applicable to most genes and tissues and includes powerful novel data annotation and visualization tools for post-transcriptional regulation.

Far from home: the role of glial mRNA localization in synaptic plasticity.

Neurons and glia are highly polarized cells, whose distal cytoplasmic functional subdomains require specific proteins. Neurons have axonal and dendritic cytoplasmic extensions containing synapses whose plasticity is regulated efficiently by mRNA transport and localized translation. The principles behind these mechanisms are equally attractive for explaining rapid local regulation of distal glial cytoplasmic projections, independent of their cell nucleus. However, in contrast to neurons, mRNA localization has received little experimental attention in glia. Nevertheless, there are many functionally diverse glial subtypes containing extensive networks of long cytoplasmic projections with likely localized regulation that influence neurons and their synapses. Moreover, glia have many other neuron-like properties, including electrical activity, secretion of gliotransmitters and calcium signaling, influencing, for example, synaptic transmission, plasticity and axon pruning. Here, we review previous studies concerning glial transcripts with important roles in influencing synaptic plasticity, focusing on a few cases involving localized translation. We discuss a variety of important questions about mRNA transport and localized translation in glia that remain to be addressed, using cutting-edge tools already available for neurons.

Super-Resolution Single Molecule FISH at the Drosophila Neuromuscular Junction.

The lack of an effective, simple, and highly sensitive protocol for fluorescent in situ hybridization (FISH) at the Drosophila larval neuromuscular junction (NMJ) has hampered the study of mRNA biology. Here, we describe our modified single molecule FISH (smFISH) methods that work well in whole mount Drosophila NMJ preparations to quantify primary transcription and count individual cytoplasmic mRNA molecules in specimens while maintaining ultrastructural preservation. The smFISH method is suitable for high-throughput sample processing and 3D image acquisition using any conventional microscopy imaging modality and is compatible with the use of antibody colabeling and transgenic fluorescent protein tags in axons, glia, synapses, and muscle cells. These attributes make the method particularly amenable to super-resolution imaging. With 3D Structured Illumination Microscopy (3D-SIM), which increases spatial resolution by a factor of 2 in X, Y, and Z, we acquire super-resolution information about the distribution of single molecules of mRNA in relation to covisualized synaptic and cellular structures. Finally, we demonstrate the use of commercial and open source software for the quality control of single transcript expression analysis, 3D-SIM data acquisition and reconstruction as well as image archiving management and presentation. Our methods now allow the detailed mechanistic and functional analysis of sparse as well as abundant mRNAs at the NMJ in their appropriate cellular context.

Drosophila sensory cilia lacking MKS proteins exhibit striking defects in development but only subtle defects in adults.

Cilia are conserved organelles that have important motility, sensory and signalling roles. The transition zone (TZ) at the base of the cilium is crucial for cilia function, and defects in several TZ proteins are associated with human congenital ciliopathies such as nephronophthisis (NPHP) and Meckel-Gruber syndrome (MKS). In several species, MKS and NPHP proteins form separate complexes that cooperate with Cep290 to assemble the TZ, but flies seem to lack core components of the NPHP module. We show that MKS proteins in flies are spatially separated from Cep290 at the TZ, and that flies mutant for individual MKS genes fail to recruit other MKS proteins to the TZ, whereas Cep290 seems to be recruited normally. Although there are abnormalities in microtubule and membrane organisation in developing MKS mutant cilia, these defects are less apparent in adults, where sensory cilia and sperm flagella seem to function quite normally. Thus, localising MKS proteins to the cilium or flagellum is not essential for viability or fertility in flies.