1. Genes That Affect The Assembly Of The Nervous System In Drosophila Embryos Identified by RNA Interference.? ? RNA interference (RNAi) has been shown to be a powerful method to study the function of genes in vivo by silencing endogenous mRNA with double-stranded (ds) RNA. Previously, we performed in vivo RNAi screening and identified 43 Drosophila genes, including 18 novel genes required for the development of the embryonic nervous system. In the present study, 22 additional genes affecting embryonic nervous system development were found. Novel RNAi-induced phenotypes affecting nervous system development were found for 16 of the 22 genes. Seven of the genes have unknown functions. Other genes found encode transcription factors, a chromatin-remodeling protein, membrane receptors, signaling molecules and proteins involved in cell adhesion, RNA binding, and ion transport. Human orthologs were identified for proteins encoded by 16 of the genes. The total number of dsRNAs that we have tested for an RNAi-induced phenotype affecting the embryonic nervous system, including our previous study, is 7,312, which corresponds to about 50% of the genes in the Drosophila genome.? ? Cultures of neuroblasts that generate abundant neurons were established from Drosophila embroys to study silencing of genes by RNA interference. Cultured cells expressed ELAV, a marker of neurons, Futsch, a marker of neurites, and Synapsin, Synaptobrevin, and Synaptogamin, proteins involved in neurotransmitter secretion. Conditions were found for efficient transfection of cells with siRNAs for ELAV or the insulin-like receptor, which resulted in marked decreases in neurons that express ELAV and Futsch. Cells also were successfully transfected with long-chain Sox-Neuro dsRNA resulting in a 55% reduction of neurons expressing Futsch. The results suggest that this cultured neural cell system can be used to study RNAi-dependent silencing of genes involved in many kinds of neural functions.? ? 2. vnd/NK-2 Homeobox Gene Regulation.? ? Expression of the ventral nervous system defective/NK-2 (vnd/NK-2) homeobox gene initiates neural development in part of the neuroectoderm of the Drosophila embryo that gives rise to some cephalic neuroblasts (NBs) and all medial and two intermediate NBs per hemisegment of the ventral nerve cord. vnd/NK-2 homeodomain protein also is required for the specification of 11 ventral nerve cord NBs per hemisegment. Each NB is a different cell type.? ? We have continued to study how a pattern of neuroblasts is generated in the CNS. Nucleotide sequences within the vnd/NK-2 5-prime-upstream enhancer (-2.8 to -5.3 kb) that were conserved during evolution were identified by comparing genomic nucleotide sequences from seven Drosophila species that diverged between 3 and 40 million years ago. Multiple highly-conserved sequences were found. DNA constructs of the vnd/NK-2 5-prime-upstream region of the vnd/NK-2 gene were prepared, each with a small deletion of a highly-conserved nucleotide sequence, and were linked to reporter genes that could be detected by immunohistochemistry. Transgenic fly lines were obtained for each DNA construct. The expression patterns of the reporter genes were analyzed by immunohistochemistry. Nucleotide sequences were identified that regulate the expression of the reporter genes in different subsets of neuroblasts. Candidate binding sites for proteins that may regulate the vnd/NK-2 gene were identified.? ? 3. Identification Of Compounds That Increase CREB Activity As Possible Enhancers Of Long-Term Memory.? ? To identify compounds that enhance activation of the CREB gene, approximately 73,000 compounds were screened, each at 7 to 24 concentrations, using a cell-based Beta-lactamase reporter gene assay in a quantitative high-throughput screening format. The assay is based upon a Beta-lactamase reporter gene under the control of CREB binding sites in DNA. We rapidly identified 1,062 compounds with half-maximal enhancement of CREB-mediated activation of gene expression ranging from 0.016 to 10 uM. Selected compounds were confirmed using a luciferase reporter gene assay. To study the mechanism of action of some of the compounds, their effects on cAMP levels, protein kinase A and phosphodiesterase activities were determined. Some of the compounds were identified as novel, potent phosphodiesterase inhibitors. The compounds identified in this screen are candidate enhancers of long-term memory via CREB activation of gene expression.? ? 4. longitudinals lacking (lola) Gene.? ? lola encodes a family of zinc-finger proteins that are generated by alternative splicing. lola is required for the longitudinal growth of some CNS axons and for pathfinding and target recognition of SNb motor neuron axons in Drosophila embroys. Lola protein also regulates midline crossing of CNS axons in the ventral nerve cord. Seventeen species of Lola protein that have the same N-terminal region but different zinc fingers in the C-terminal regions are formed through alternative mRNA splicing. We tested the hypothesis that isoforms of Lola protein that contain different zinc fingers regulate different sets of genes. Double-stranded RNAs targeting the constant N-terminal region or sequences within the variable C-terminal regions of lola mRNA, excluding the zinc finger regions, were synthesized, and each double-stranded RNA was injected into early Drosophila embryos to decrease the level of the corresponding species of lola mRNA by RNA interference. RNAs were purified from injected embryos that had been incubated. Biotin-labeled cRNAs were prepared and used for hybridization to Drosophila genomic microarrays. The results show that Lola proteins are transcription factors that, directly or indirectly, regulate gene expression, and that some of the isoforms of lola mRNA encode proteins that regulate different sets of genes.? ? 5. Effect Of An Ecdysone Analog, Muristerone A, on a Drosophila Neural Cell Line.? ? The effects of the ecdysone analog, muristerone A, over time on the levels of mRNA of cells from a neural cell line were compared to those in a non-neural cell line (Schneider's S2 cells). Five hundred and fifty-six genes were identified that are uniquely expressed by the neural cells. Exposure of the neural cells to muristerone A resulted in increases or decreases in the levels of 1,936 RNA transcripts. One hundred and forty-nine and five hundred and sixty-four mRNAs were identified that are up- or down-regulated by muristerone A only in the neural cells after 5 or 48 hours, respectively.? ? 6. Neuroligins.? ? In vertebrates, binding of neuroligins, which are post-synaptic cell adhesion proteins, to the pre-synaptic Beta-neurexin protein modulates the formation, composition, and functional properties of synapses. The Drosophila genome contains four genes encoding putative neuroligins. The expression of each neuroligin gene was determined at different stages of the Drosophila life cycle. Neuroligin 1 was only present in adult brain, and was specifically localized to part of the mushroom body, the anatomical site of odor-mediated associative learning in the fly. A deletion mutation of the neuroligin 1 gene was generated that removed the entire coding region and 22 kb of 5-prime regulatory sequences. In neuroligin 1 null mutants olfactory acuity and response to electric shock was normal, but when tested in an olfactory conditioning paradigm, mutant flies exhibited a marked reduction in associative learning compared to wild-type controls when octanol was used as the condition stimulus, but relatively normal learning was observed in response to methyl cyclohexanol. These results suggest that associative learning involving octanol and electric shock in Drosophila may be mediated in part by neuroligin 1 dependent regulation of some synapses in the mushroom body.
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