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Wesle-affy-droso-91201

ABSTRACT: Neurogenesis in all animals is dependent on the function of different Notch receptors and their ligands. Drosophila is an excellent system to understand the principles and mechanisms governing this conserved process. Since the different Notch receptors and their ligands are expressed and function in the same cells, or overlapping stages of neurogenesis, and since there is extensive feedback regulation between them, it is very difficult to identify the target genes of each receptor or ligand in vivo. We have developed an excellent model system based on Drosophila cultured Schneider (S2) cells that faithfully represents in vivo activities. We propose microarray analysis with this system to identify the suite of putative target genes that are independently regulated by each protein in our study. We also expect to gather information about the signaling pathways used by the different receptors and their ligands, and develop hypotheses for testing during neurogenesis in vivo. These efforts will contribute significantly towards achieving the Specific Objectives of the grant parent (R01 NS043122, ‘Different Notch Receptors in Drosophila Development’). To determine the sets of genes regulated by NFull, NDCterm, and Delta through microarray analyses of RNA extracted from S2 cells expressing these proteins that were treated or not treated with S2 cells expressing their ligand or receptor. Neurogenesis in Drosophila initiates within clusters of cells that have acquired the potential to become neuronal cells. The action of the full-length Notch receptor, NFull, and the ligand Delta make the majority of these cells to differentiate the epidermis. The action of a truncated Notch receptor, NDCterm, and Delta make the remaining cells to differentiate the nervous system. We have recently shown that Delta also has activities independent of Notch receptors that promote neurogenesis. We will test the hypothesis that NFull would promote expression of epidermal differentiation genes while NDCterm and Delta that would promote expression of neuronal differentiation genes. We will also test the hypothesis that genes regulated by NDCterm and Delta would be the same or would complement each other’s activities. We request the Microarray Consortium to process our samples and help us analyze the data. We will select a set of genes for validation by northern blotting or QPCR on similar samples of RNA from S2 cells and in vivo materials. We have available in the lab fly stocks carrying mutants (nulls) alleles or inducible transgenes for all the proteins used in this microarray experiment. Funds from the parent grant will be used for these in vitro and in vivo validation experiments. This project proposal is a part of the Supplementary Proposal approved by NIH/NINDS. Project proposals covering other parts, the effects on secreted ligands of Scabrous and Wingless, will be submitted when we have ready the RNAs from these experiments. Keywords: other

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE2863 | GEO | 2005/07/06

SECONDARY ACCESSION(S): PRJNA92527

REPOSITORIES: GEO

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Project description:Neurogenesis in all animals is dependent on the function of different Notch receptors and their ligands. Drosophila is an excellent system to understand the principles and mechanisms governing this conserved process. Since the different Notch receptors and their ligands are expressed and function in the same cells, or overlapping stages of neurogenesis, and since there is extensive feedback regulation between them, it is very difficult to identify the target genes of each receptor or ligand in vivo. We have developed an excellent model system based on Drosophila cultured Schneider (S2) cells that faithfully represents in vivo activities. We propose microarray analysis with this system to identify the suite of putative target genes that are independently regulated by each protein in our study. We also expect to gather information about the signaling pathways used by the different receptors and their ligands, and develop hypotheses for testing during neurogenesis in vivo. These efforts will contribute significantly towards achieving the Specific Objectives of the grant parent (R01 NS043122,Different Notch Receptors in Drosophila Development).,To determine the sets of genes regulated by NFull, NDCterm, and Delta through microarray analyses of RNA extracted from S2 cells expressing these proteins that were treated or not treated with S2 cells expressing their ligand or receptor. ,Neurogenesis in Drosophila initiates within clusters of cells that have acquired the potential to become neuronal cells. The action of the full-length Notch receptor, NFull, and the ligand Delta make the majority of these cells to differentiate the epidermis. The action of a truncated Notch receptor, NDCterm, and Delta make the remaining cells to differentiate the nervous system. We have recently shown that Delta also has activities independent of Notch receptors that promote neurogenesis. We will test the hypothesis that NFull would promote expression of epidermal differentiation genes while NDCterm and Delta that would promote expression of neuronal differentiation genes. We will also test the hypothesis that genes regulated by NDCterm and Delta would be the same or would complement each otherâs activities.,We request the Microarray Consortium to process our samples and help us analyze the data. We will select a set of genes for validation by northern blotting or QPCR on similar samples of RNA from S2 cells and in vivo materials. We have available in the lab fly stocks carrying mutants (nulls) alleles or inducible transgenes for all the proteins used in this microarray experiment. Funds from the parent grant will be used for these in vitro and in vivo validation experiments. This project proposal is a part of the Supplementary Proposal approved by NIH/NINDS. Project proposals covering other parts, the effects on secreted ligands of Scabrous and Wingless, will be submitted when we have ready the RNAs from these experiments.

Project description:BACKGROUND: The Notch signaling pathway is an evolutionary conserved signal transduction pathway involved in embryonic patterning and regulation of cell fates during development and self-renewal. Recent studies have demonstrated that this pathway is integral to a complex system of interactions, involving as well other signal transduction pathways, and implicated in distinct human diseases. Delta-like 1 (Dll1) is one of the known ligands of the Notch receptors. The role of the Notch ligands is less well understood. Loss-of-function of Dll1 leads to embryonic lethality, but reduction of Delta-like 1 protein levels has not been studied in adult stage. METHODOLOGY/PRINCIPAL FINDINGS: Here we present the haploinsufficient phenotype of Dll1 and a missense mutant Dll1 allele (Dll1(C413Y)). Haploinsufficiency leads to a complex phenotype with several biological processes altered. These alterations reveal the importance of Dll1 mainly in metabolism, energy balance and in immunology. The animals are smaller, lighter, with altered fat to lean ratio and have increased blood pressure and a slight bradycardia. The animals have reduced cholesterol and triglyceride levels in blood. At the immunological level a subtle phenotype is observed due to the effect and fine-tuning of the signaling network at the different levels of differentiation, proliferation and function of lymphocytes. Moreover, the importance of the proteolytic regulation of the Notch signaling network emphasized. CONCLUSIONS/SIGNIFICANCE: In conclusion, slight alterations in one player of Notch signaling alter the entire organism, emphasizing the fine-tuning character of this pathway in a high number of processes. Four organs (liver, spleen, thymus, brain) of the Dll1 mutant mouse line analysed by cDNA microarray technology. Experiments include four biological replicates for reference (wildtype) and mutant animals. Two technical replicates for each mutant mouse were performed. As reference pooled RNA of the same organ was used. 50% of the chip hybridisations are dye sway experiments.

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Wesle-affy-droso-91201

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