Visualizing adenosine-to-inosine RNA editing in the Drosophila nervous system

Informational recoding by adenosine-to-inosine RNA editing diversifies neuronal proteomes by chemically modifying structured mRNAs. However, techniques for analyzing editing activity on substrates in defined neurons in vivo are lacking. Guided by comparative genomics, here we reverse-engineered a fluorescent reporter sensitive to Drosophila melanogaster adenosine deaminase that acts on RNA (dADAR) activity and alterations in dADAR autoregulation. Using this artificial dADAR substrate, we visualized variable patterns of RNA-editing activity in the Drosophila nervous system between individuals. Our results demonstrate the feasibility of structurally mimicking ADAR substrates as a method to regulate protein expression and, potentially, therapeutically repair mutant mRNAs. Our data suggest variable RNA editing as a credible molecular mechanism for mediating individual-to-individual variation in neuronal physiology and behavior.     

Drosophila stathmin: a microtubule-destabilizing factor involved in nervous system formation

Stathmin is a ubiquitous regulatory phosphoprotein, the generic element of a family of neural phosphoproteins in vertebrates that possess the capacity to bind tubulin and interfere with microtubule dynamics. Although stathmin and the other proteins of the family have been associated with numerous cell regulations, their biological roles remain elusive, as in particular inactivation of the stathmin gene in the mouse resulted in no clear deleterious phenotype. We identified stathmin phosphoproteins in Drosophila, encoded by a unique gene sharing the intron/exon structure of the vertebrate stathmin and stathmin family genes. They interfere with microtubule assembly in vitro, and in vivo when expressed in HeLa cells. Drosophila stathmin expression is regulated during embryogenesis: it is high in the migrating germ cells and in the central and peripheral nervous systems, a pattern resembling that of mammalian stathmin. Furthermore, RNA interference inactivation of Drosophila stathmin expression resulted in germ cell migration arrest at stage 14. It also induced important anomalies in nervous system development, such as loss of commissures and longitudinal connectives in the ventral cord, or abnormal chordotonal neuron organization. In conclusion, a single Drosophila gene encodes phosphoproteins homologous to the entire vertebrate stathmin family. We demonstrate for the first time their direct involvement in major biological processes such as development of the reproductive and nervous systems.     

Genes involved in the development of the peripheral nervous system of Drosophila

The development of the peripheral nervous system (PNS) requires the activity of a number of genes. The neurogenic and the proneural genes are necessary in the earliest phase; their mutations lead to hyperplasia and partial or total elimination of the PNS respectively. Some of these mutations also affect other developmental processes. Other mutations affect later events: cut transforms one type of sensory organ into another; numb alters the fate of the components of a single sensory organ. We will describe the effects of the best studied mutations on PNS development and discuss the possible role of the wild type genes.     

LAMMER kinase Kic1 is involved in pre-mRNA processing

The LAMMER kinases are conserved through evolution. They play vital roles in cell growth/differentiation, development, and metabolism. One of the best known functions of the kinases in animal cells is the regulation of pre-mRNA splicing. Kic1 is the LAMMER kinase in fission yeast Schizosaccharomyces pombe. Despite the reported pleiotropic effects of kic1⁺ deletion/overexpression on various cellular processes the involvement of Kic1 in splicing remains elusive. In this study, we demonstrate for the first time that Kic1 not only is required for efficient splicing but also affects mRNA export, providing evidence for the conserved roles of LAMMER kinases in the unicellular context of fission yeast. Consistent with the hypothesis of its direct participation in multiple steps of pre-mRNA processing, Kic1 is predominantly present in the nucleus during interphase. In addition, the kinase activity of Kic1 plays a role in modulating its own cellular partitioning. Interestingly, Kic1 expression oscillates in a cell cycle-dependent manner and the peak level coincides with mitosis and cytokinesis, revealing a potential mechanism for controlling the kinase activity during the cell cycle. The novel information about the in vivo functions and regulation of Kic1 offers insights into the conserved biological roles fundamental to LAMMER kinases in eukaryotes.     

The sympathetic nervous system is involved in variant Creutzfeldt-Jakob disease

Prion epizoonoses spread from animals consumed by humans raise the question of which pathways lead to prion neuroinvasion after oral exposure of humans. Here we show that neurons of sympathetic ganglia of patients with variant Creutzfeldt-Jakob disease (vCJD) accumulate the abnormal isoform of the protein prion. This observation shows the involvement of the sympathetic nervous system in the pathogenesis of vCJD and suggests a role for GUT-associated sympathetic neurons in prion propagation in humans after oral contamination.     

Clathrin-coated vesicles in nervous tissue are involved primarily in synaptic vesicle recycling

The recycling of synaptic vesicles in nerve terminals is thought to involve clathrin-coated vesicles. However, the properties of nerve terminal coated vesicles have not been characterized. Starting from a preparation of purified nerve terminals obtained from rat brain, we isolated clathrin-coated vesicles by a series of differential and density gradient centrifugation steps. The enrichment of coated vesicles during fractionation was monitored by EM. The final fraction consisted of greater than 90% of coated vesicles, with only negligible contamination by synaptic vesicles. Control experiments revealed that the contribution by coated vesicles derived from the axo-dendritic region or from nonneuronal cells is minimal. The membrane composition of nerve terminal-derived coated vesicles was very similar to that of synaptic vesicles, containing the membrane proteins synaptophysin, synaptotagmin, p29, synaptobrevin and the 116-kD subunit of the vacuolar proton pump, in similar stoichiometric ratios. The small GTP-binding protein rab3A was absent, probably reflecting its dissociation from synaptic vesicles during endocytosis. Immunogold EM revealed that virtually all coated vesicles carried synaptic vesicle proteins, demonstrating that the contribution by coated vesicles derived from other membrane traffic pathways is negligible. Coated vesicles isolated from the whole brain exhibited a similar composition, most of them carrying synaptic vesicle proteins. This indicates that in nervous tissue, coated vesicles function predominantly in the synaptic vesicle pathway. Nerve terminal-derived coated vesicles contained AP-2 adaptor complexes, which is in agreement with their plasmalemmal origin. Furthermore, the neuron-specific coat proteins AP 180 and auxilin, as well as the alpha a1 and alpha c1-adaptins, were enriched in this fraction, suggesting a function for these coat proteins in synaptic vesicle recycling.     

Notch signaling is involved in nervous system formation in ascidian embryos

Notch signaling plays crucial roles during embryogenesis in various metazoans. HrNotch, a Notch homologue in the ascidian Halocynthia roretzi, has been previously cloned, and its expression pattern suggests that HrNotch signaling is involved in nervous system formation. To determine the function of HrNotch signaling, in the present study we examined the effects of the constitutively activated forms of HrNotch. Overexpression resulted in larvae with defects in neural tube closure and brain vesicle formation. In embryos expressing the activated HrNotch, the expression of a neural marker gene, HrETR-1, was enhanced and expanded in the central nervous system, although ectopic expression decreased during the tailbud stage. The activated HrNotch also suppressed the formation of the adhesive organ (palps) and the peripheral nervous system, which consists of ciliary mechanosensory neurons, whereas it promoted epidermal differentiation. The suppression and promotion of the formation of these respective cell types were confirmed by examination of the expression of relevant tissue-specific markers. We also cloned Hrdelta, an ascidian homologue of DSL family genes, which encode ligands for which Notch acts as a receptor. The expression of Hrdelta was observed in the precursors of palps and peripheral neurons in addition to the CNS. These results suggest that Notch signaling is important for ascidian nervous system formation and that it affects the fate choice between palps and epidermis and between peripheral neurons and epidermis within the neurogenic regions of the surface ectoderm by suppressing the formations of palps and peripheral neurons and promoting epidermal differentiation.     

Genetic analysis of vein function in the Drosophila embryonic nervous system.

The Drosophila epidermal growth factor receptor (EGFR) may be activated by two ligands expressed in the embryonic nervous system, Spitz and Vein. Previous studies have established Spitz as an essential activator of EGFR signaling in nervous system development. Here, we report the pattern of expression of vein mRNA in the nervous system and characterize the contribution of vein to cell lineage and axonogenesis. The number of midline glia (MG) precursors is reduced in vein mutants before the onset of embryonic apoptosis. In contrast to spitz, mis-expression of vein does not suppress apoptosis in the MG. These data indicate that early midline EGFR signaling, requiring vein and spitz, establishes MG precursor number, whereas later EGFR signals, requiring spitz, suppress apoptosis in the MG. vein mutants show early irregularities during axon tract establishment, which resolve later to variable defasciculation and thinner intersegmental axon tracts. vein and spitz phenotypes act additively in the regulation of MG cell number, but show synergism in a midline neuronal cell number phenotype and in axon tract architecture. vein appears to act downstream of spitz to briefly amplify local EGFR activation.     

BCAS2 is essential for Drosophila viability and functions in pre-mRNA splicing

Here, we show that dBCAS2 (CG4980, human Breast Carcinoma Amplified Sequence 2 ortholog) is essential for the viability of Drosophila melanogaster. We find that ubiquitous or tissue-specific depletion of dBCAS2 leads to larval lethality, wing deformities, impaired splicing, and apoptosis. More importantly, overexpression of hBCAS2 rescues these defects. Furthermore, the C-terminal coiled-coil domain of hBCAS2 binds directly to CDC5L and recruits hPrp19/PLRG1 to form a core complex for splicing in mammalian cells and can partially restore wing damage induced by knocking down dBCAS2 in flies. In summary, Drosophila and human BCAS2 share a similar function in RNA splicing, which affects cell viability.     

TRIM9 is specifically expressed in the embryonic and adult nervous system

The TRIM family members are defined by the presence of the tripartite motif (RING, B-box and coiled-coil domains or RBCC). They have been implicated in a variety of processes, such as regulation of development and oncogenesis. We report the expression analysis of a member of this family, TRIM9. Its expression is mainly confined to the central nervous system. The developing neocortex, the dorsal thalamus, the midbrain, the basal area of the hindbrain and spinal cord show high level of expression during embryogenesis. In adult brain, TRIM9 is detected in the Purkinje cells of the cerebellum, in the hippocampus, and in the cortex.     

An altered form of pp60c-src is expressed primarily in the central nervous system.

The expression of two forms of pp60c-src, pp60 and pp60+, was measured in the central nervous system (CNS) and the peripheral nervous system. Both forms were expressed in the CNS, whereas only pp60 was primarily detected in the peripheral nervous system. Our findings suggest that pp60+ may play a role in events important to the CNS.     

Integration of complex larval chemosensory organs into the adult nervous system of Drosophila

The sense organs of adult Drosophila, and holometabolous insects in general, derive essentially from imaginal discs and hence are adult specific. Experimental evidence presented here, however, suggests a different developmental design for the three largely gustatory sense organs located along the pharynx. In a comprehensive cellular analysis, we show that the posteriormost of the three organs derives directly from a similar larval organ and that the two other organs arise by splitting of a second larval organ. Interestingly, these two larval organs persist despite extensive reorganization of the pharynx. Thus, most of the neurons of the three adult organs are surviving larval neurons. However, the anterior organ includes some sensilla that are generated during pupal stages. Also, we observe apoptosis in a third larval pharyngeal organ. Hence, our experimental data show for the first time the integration of complex, fully differentiated larval sense organs into the nervous system of the adult fly and demonstrate the embryonic origin of their neurons. Moreover, they identify metamorphosis of this sensory system as a complex process involving neuronal persistence, generation of additional neurons and neuronal death. Our conclusions are based on combined analysis of reporter expression from P[GAL4] driver lines, horseradish peroxidase injections into blastoderm stage embryos, cell labeling via heat-shock-induced flip-out in the embryo, bromodeoxyuridine birth dating and staining for programmed cell death. They challenge the general view that sense organs are replaced during metamorphosis.