1. Genes That Affect The Development and/or Function Of The Nervous System by RNA Interference.? ? Previously, we performed in vivo RNA-i screening of about 50 percent of the genes in the Drosophila genome and identified 65 novel genes required for the development of the embryonic nervous system. During the past year we have extended our screen by injecting additional dsRNAs in embryos, and have identified additional genes with putative roles in the development of the Drosophila embryonic nervous system. ? ? A neural cell line derived from the central nervous system of Drosophila third instar larvae, ML-Dm-Bg2c2, and mouse NS26 neuroblastoma cells were used to establish tissue culture-based assays for neuronal differentiation and function. Conditions also were established to promote neurite extension in ML-Dm-Bg2c2 cell cultures. ML-Dm-Bg2c2 cells that express the reporter genes for either Luciferase, eGFP, dsRed , or SEAP under the control of a viral, or a neural-specific promoter were generated and cloned. Additionally, ML-Dm-Bg2c2 cells that express a synaptic vesicle function sensor or a Ca++ sensor were cloned.? ? 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, all ventral nerve cord medial neuroblasts, and two intermediate neuroblasts per hemisegment. Vnd/NK-2 homeodomain protein also is required to determine the cell types of the 11 ventral nerve cord neuroblasts per hemisegment. Each neuroblast is a different cell type.? ? Nucleotide sequences within the vnd/NK-2 5-prime-upstream enhancer that have been conserved during evolution were identified by comparing genomic nucleotide sequences from seven Drosophila species that diverged between 3 and 40 million years ago. Twelve 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. 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 regulate the vnd/NK-2 gene were identified. Each of the 8 types of neuroblasts studied requires a different combinatorial set of transcription factors for the expression of the vnd/NK-2 gene. Furthermore, transcription factors for 7 of the 8 neuroblasts exhibit cooperativity; i.e., omission of only one of the multiple DNA binding sites for transcription factors required for vnd/NK-2 expression in a subset of neuroblasts results in no vnd/NK-2 gene expression in those neuroblasts.? ? 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 15 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 identified 1,800 compounds with half-maximal enhancement of CREB-mediated activation of gene expression ranging from 0.016 to 100 uM. Selected compounds were confirmed using a luciferase reporter gene assay. The mechanisms of action of some compounds were studied by determining their effects on cAMP levels, protein kinase A activity, and phosphodiesterase activity. Several novel classes of phosphodiesterase inhibitors were found. Seventy seven derivatives of one of these inhibitors were synthesized and a novel highly potent, highly specific phosphodiesterase 4 inhibitor was found. The compounds identified in this screen are candidate enhancers of long-term memory via CREB activation of gene expression.We hope that eventually one or more of the compounds found may be useful therapeutically to enhance long-term memory in Alzheimers patients.? ? ? 4. longitudinals lacking (lola) Gene.? ? The Drosophila gene lola encodes a family of at least 20 proteins that are formed by alternative splicing of RNA that are involved in axon and dendrite pathfinding and wiring specificity. All of the Lola proteins have an identical N-terminal region that contains a BTB protein dimerization domain, but each has a different C-terminal region. Seventeen species of Lola protein have different zinc fingers in the C-terminal regions. We tested the hypothesis that isoforms of Lola protein that contain different zinc fingers regulate different sets of genes. Double-stranded RNAs corresponding to the constant N-terminal region of Lola proteins or to part of several variable C-terminal isoforms of Lola proteins were synthesized and each RNA was injected into early Drosophila embryos to decrease the level of the corresponding species of Lola mRNA by RNA interference. The effects of RNA interference on gene expression were determined by hybridization of RNA to nucleic acid microarrays. Injection of double-stranded RNA targeting the constant N-terminal region of Lola found in all Lola protein isoforms resulted in changes in the expression of 206 genes; i.e., repression of 147 genes and induction of 59 genes. The results show that Lola proteins are transcription factors that, directly or indirectly, regulate gene expression, and that two 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.? ? Exposure of the neural cells to muristerone A resulted in changes in the levels of 1,936 RNA transcripts, including one hundred and forty-nine and five hundred and sixty-four mRNAs that are up- or down-regulated, respectively, by muristerone A in the neural cells, but not in non-neural Schneider cells. Seven miRNAs were found whose levels of expression are increased or decreased in ML-Dm-Bg2c2 cells after 5 to 48 hours exposure to muristerone A, but not in cells pre-treated with Ecdysone-Receptor siRNA.? ? 6. Neuroligins.? ? An inducible transgene containing the wildtype fnrlg1 gene was constructed using RT-PCR clones isolated using adult head RNA from the w;CS strain. In the fnrlg1 null genetic background, expression of wildtype fnrlg in mushroom body neurons increased learning as measured using the olfactory associative learning assay. Three fnrlg1:gfp transgenes were cloned that expressed native membrane localized fluorescent protein in a Drosophila neuronal cell line. Transgenic fly strains containing each of the three GFP fusions were obtained and expression of the transgenes were induced by crossing the lines to a fnrlg1 promotor gal4 driver line which confers expression to adult mushroom body neurons. Confocal microscopy revealed strong labeling of axon fiber tracts in the mushroom body and the absence of labeling in the dendrites. This localization suggests that fnrlg1:gfp may be presynaptic in mushroom body neurons.
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