Loss of embryonic ion channel function leads to morphological defects, but the underlying reason for these defects remains elusive. Here, we show that inwardly rectifying potassium (Irk) channels regulate release of the bone morphogenetic protein Dpp in the developing fly wing and that this is necessary for developmental signaling. Inhibition of Irk channels decreases the incidence of distinct Dpp-GFP release events above baseline fluorescence while leading to a broader distribution of Dpp-GFP. Work by others in different cell types has shown that Irk channels regulate peptide release by modulating membrane potential and calcium levels. We found calcium transients in the developing wing, and inhibition of Irk channels reduces the duration and amplitude of calcium transients. Depolarization with high extracellular potassium evokes Dpp release. Taken together, our data implicate Irk channels as a requirement for regulated release of Dpp, highlighting the importance of the temporal pattern of Dpp presentation for morphogenesis of the wing.

During morphogenesis, cells communicate with each other to shape tissues and organs. Several lines of recent evidence indicate that ion channels play a key role in cellular signaling and tissue morphogenesis. However, little is known about the scope of specific ion-channel types that impinge upon developmental pathways. The Drosophila melanogaster wing is an excellent model in which to address this problem as wing vein patterning is acutely sensitive to changes in developmental pathways. We conducted a screen of 180 ion channels expressed in the wing using loss-of-function mutant and RNAi lines. Here we identify 44 candidates that significantly impacted development of the Drosophila melanogaster wing. Calcium, sodium, potassium, chloride, and ligand-gated cation channels were all identified in our screen, suggesting that a wide variety of ion channel types are important for development. Ion channels belonging to the pickpocket family, the ionotropic receptor family, and the bestrophin family were highly represented among the candidates of our screen. Seven new ion channels with human orthologs that have been implicated in human channelopathies were also identified. Many of the human orthologs of the channels identified in our screen are targets of common general anesthetics, anti-seizure and anti-hypertension drugs, as well as alcohol and nicotine. Our results confirm the importance of ion channels in morphogenesis and identify a number of ion channels that will provide the basis for future studies to understand the role of ion channels in development.

It is widely accepted that long-term changes in synapse structure and function are mediated by rapid activity-dependent gene transcription and new protein synthesis. A growing amount of evidence suggests that the microRNA (miRNA) pathway plays an important role in coordinating these processes. Despite recent advances in this field, there remains a critical need to identify specific activity-regulated miRNAs as well as their key messenger RNA (mRNA) targets. To address these questions, we used the larval Drosophila melanogaster neuromuscular junction (NMJ) as a model synapse in which to identify novel miRNA-mediated mechanisms that control activity-dependent synaptic growth. First, we developed a screen to identify miRNAs differentially regulated in the larval CNS following spaced synaptic stimulation. Surprisingly, we identified five miRNAs (miRs-1, -8, -289, -314, and -958) that were significantly downregulated by activity. Neuronal misexpression of three miRNAs (miRs-8, -289, and -958) suppressed activity-dependent synaptic growth suggesting that these miRNAs control the translation of biologically relevant target mRNAs. Functional annotation cluster analysis revealed that putative targets of miRs-8 and -289 are significantly enriched in clusters involved in the control of neuronal processes including axon development, pathfinding, and growth. In support of this, miR-8 regulated the expression of a wingless 3′UTR (wg 3′ untranslated region) reporter in vitro. Wg is an important presynaptic regulatory protein required for activity-dependent axon terminal growth at the fly NMJ. In conclusion, our results are consistent with a model where key activity-regulated miRNAs are required to coordinate the expression of genes involved in activity-dependent synaptogenesis.

SummaryThe temporal and spatial regulation of protein synthesis plays an important role in the control of neural physiology. In axons and dendrites, translationally repressed mRNAs are actively transported to their destinations in a variety of ribonucleoprotein particles (RNPs). A subset of these neuronal RNPs has been shown to contain proteins associated with mRNA processing bodies (P bodies). P bodies are a class of highly conserved cytoplasmic granules that have been linked to both mRNA decay and translational repression via general and miRNA-mediated pathways. Here, we characterize functions for HPat/Pat1 (also known as Patr-1), a core component of P bodies, at the glutamatergic larval Drosophila neuromuscular junction (NMJ). We show that hpat mutants exhibit a strong synaptic hyperplasia at the NMJ. The synaptic defects observed in hpat mutants are associated with rearrangement of the axonal microtubule cytoskeleton suggesting that HPat negatively regulates presynaptic microtubule-based growth during NMJ development. Consistent with this, overexpression of HPat also blocks the rapid growth of presynaptic boutons induced by spaced depolarization. Finally, we demonstrate that HPat interacts genetically with the catalytic subunit of the deadenylase complex (twin/CCR4) and the miRNA pathway (Argonaute 1) to control bouton formation. We propose that HPat is required to target mRNAs involved in the control of microtubule architecture and synaptic terminal growth for repression, presumably in P bodies, via both general and miRNA-mediated mechanisms.

The exonuclease torpedo Xrn2 loads onto nascent RNA 5′-PO4ends and chases down pol II to promote termination downstream from polyA sites. We report that Xrn2 is recruited to preinitiation complexes and “travels” to 3′ ends of genes. Mapping of 5′-PO4ends in nascent RNA identified Xrn2 loading sites stabilized by an active site mutant, Xrn2(D235A). Xrn2 loading sites are approximately two to 20 bases downstream from where CPSF73 cleaves at polyA sites and histone 3′ ends. We propose that processing of all mRNA 3′ ends comprises cleavage and limited 5′–3′ trimming by CPSF73, followed by handoff to Xrn2. A similar handoff occurs at tRNA 3′ ends, where cotranscriptional RNase Z cleavage generates novel Xrn2 substrates. Exonuclease-dead Xrn2 increased transcription in 3′ flanking regions by inhibiting polyA site-dependent termination. Surprisingly, the mutant Xrn2 also rescued transcription in promoter-proximal regions to the same extent as in 3′ flanking regions. eNET-seq revealed Xrn2-mediated degradation of sense and antisense nascent RNA within a few bases of the TSS, where 5′-PO4ends may be generated by decapping or endonucleolytic cleavage. These results suggest that a major fraction of pol II complexes terminates prematurely close to the start site under normal conditions by an Xrn2-mediated torpedo mechanism.

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability and is caused by mutations in the gene encoding for the Fragile X messenger ribonucleoprotein (FMRP). FMRP is an evolutionarily conserved and neuronally enriched RNA binding protein (RBP) with functions in the control of processes including RNA editing, RNA transport, and protein translation. Specific target RNAs play critical roles in neurodevelopment including the regulation of neurite morphogenesis, synaptic plasticity, and cognitive function. The different biological functions of FMRP are modulated by its cooperative interaction with distinct sets of neuronal RNA and protein binding partners. Here, we focus on interactions between FMRP and components of the microRNA (miRNA) pathway. Using the Drosophila model system, we show that dFMRP can repress the translation of a reporter mRNA via a deadenylation-independent mechanism. This repression requires the activity of both AGO1 and GW182, conserved components of the miRNA-containing RISC (miRISC). Interestingly, we find that dFMRP can bind directly to a short stem loop structure in the reporter and that dFMRP binding is a prerequisite for repression by miR-958. Finally, we show that dFmr1 interacts genetically with GW182 to control neurite morphogenesis. Collectively, these data suggest the dFMRP can directly recruit the miRISC to nearby miRNA binding sites and then repress translation via the activity of the miRISC effector, GW182.