Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of Drosophila model of mitochondrial disease

ABSTRACT: Background: A point mutation in the Drosophila gene technical knockout (tko), encoding mitoribosomal protein S12, provokes a phenotype of respiratory chain deficiency, developmental delay and neurological abnormalities similar to those presented in many human mitochondrial disorders, as well as defective courtship behaviour. Results: Transcriptome-wide analysis of gene expression in tko25t mutant flies revealed systematic, compensatory changes in expression of genes connected with metabolism, including upregulation of lactate dehydrogenase and of many genes involved in the catabolism of fats and proteins, the TCA cycle and anaplerotic pathways feeding into it. Gut-specific enzymes involved in the primary mobilization of dietary fat and protein, as well as a number of transport functions, were strongly upregulated, consistent with the idea that OXPHOS dysfunction is perceived physiologically as a starvation for particular biomolecules. Many stress-response genes were also induced. Other changes may reflect a signature of developmental delay. There was also down-regulation of genes connected with reproduction, including gametogenesis, especially in females, and courtship behaviour in males. This might represent a programmed response to a mitochondrially generated starvation signal. Although human sexual behaviour is not known to respond to mitochondrial dysfunction, the underlying signaling pathway, if conserved, could influence many physiological processes in response to nutritional stress. Transcriptomic analysis suggested the possible involvement of the Akt1/sgg signaling pathway(s). Conclusions: The transformation of metabolism in response to mitochondrial dysfunction, including digestive and absorptive functions, gives important clues as to how novel therapeutic strategies for mitochondrial disorders might be developed. Experiment Overall Design: Crosses and maintenance: Inbreeding under stressful conditions inevitably results in the selection of compensatory alleles of many genes. In order to avoid such issues, and thus determine the global effects on gene expression of the tko25t mutation in a truly unselected and 'wild-type' background, we outbred tko25t over more than 10 generations by back-crossing to each of two commonly used wild-type strains, Canton S and Oregon R. Subsequent to this backcrossing, tko25t was maintained in each background using a balancer chromosome. These stocks were then used to generate a tko25t mutant F1 generation, by crossing virgin Canton S tko25t homozygous mutant females with Oregon R tko25t males. For comparison, we generated otherwise isogenic wild-type F1 progeny by crossing virgin Canton S wild-type females with Oregon R wild-type males. Experiment Overall Design: Samples: Both tko25t and wild-type Drosophila adult flies of both sexes were collected and aged to 1 day old prior to RNA extraction and hybridization on Affymetrix microarrays. We extracted RNA from 3 independent such crosses. Experiment Overall Design: Comparative analysis: Data analysis for each gene in the array compared each tko25t mutant RNA isolate with each wild-type RNA isolate from flies of the given sex, generating 9 comparative measurements, over which the statistical analysis was performed.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Daniel Jose Moreno Fernandez-Ayala

PROVIDER: E-GEOD-10169 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Gene expression in a Drosophila model of mitochondrial disease.

Fernández-Ayala Daniel J M DJ Chen Shanjun S Kemppainen Esko E O'Dell Kevin M C KM Jacobs Howard T HT

PloS one 20100106 1

<h4>Background</h4>A point mutation in the Drosophila gene technical knockout (tko), encoding mitoribosomal protein S12, was previously shown to cause a phenotype of respiratory chain deficiency, developmental delay, and neurological abnormalities similar to those presented in many human mitochondrial disorders, as well as defective courtship behavior.<h4>Methodology/principal findings</h4>Here, we describe a transcriptome-wide analysis of gene expression in tko(25t) mutant flies that revealed s ...[more]

PMID: 20066047

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Project description:Disruption of learning and memory can occur for a number of reasons including chronic alcohol abuse, head injury, anoxia, and various neurodegenerative disorders such as Alzheimer's disease. An understanding of the basic mechanisms underlying learning and memory is essential for prevention and treatment of such brain disorders. Recent studies unequivocally demonstrated the astonishing similarities in the basic mechanisms of learning and memory between divergent species, indicating that studies of experimentally tractable model organisms with relatively simpler nervous systems are likely to produce valuable information that will facilitate our understanding of learning and memory in higher organisms. We are studying the experience-dependent modification in Drosophila male courtship as a physiologically relevant model of learning and memory. The genome-scale gene-profiling analysis in this model system should provide us the basic information about learning-induced changes in gene expression, which will be the foundation of our future studies to identify genes and genetic pathways specifically involved in the formation of long-term memory. The proposed gene-profiling experiment is designed to identify candidate genes and genetic pathways involved in the formation of long-term memory in the Drosophila courtship conditioning. The specific aims of this project are to: 1) compare gene expression profiles between the naive and trained wild type males using the GeneChip Drosophila Genome 2.0 Arrays and identify genes that are up- or down-regulated in the fly head immediately or 24-hr after the male fly is exposed to the condition where the long-term courtship memory is established (i.e., paired with a mated female for 7 hrs), 2) carry out the same analysis for the per null mutant males, 3) compare the identified genes between the 0 and 24-hr after training as well as the wild type and per mutant males, 4) identify candidate genes which might be involved in the long-term memory formation either upstream or downstream of per, 5) classify the candidate genes according to their putative cellular function and discern the possible genetic pathways necessary for the long-term memory formation. When a male fly is presented with a previously mated female, he will initially court her vigorously but his courtship activity is reduced over time. Subsequently, when paired with a virgin female, the male will display a reduced amount of courtship compared with naÃ¯ve males with no prior courtship experience. This modification of courtship is called âcourtship conditioningâ. We found that this type of courtship memory lasts at least 5 days when a male is trained with a mated female for 7 hrs. We have also found that the period gene (per) that is involved in circadian rhythm regulation plays a critical role in the formation of long-term, but not short-term, courtship memory. Our hypotheses are 1) the long-lasting memory in 7 h-trained males is accompanied with characteristic temporal changes in gene expression in the brain, 2) per mutant males are defective in regulation of critical genes involved in long-term courtship memory, 3) the memoryârelated changes in gene expression can be detected by comparing total head transcript levels between trained and naÃ¯ve as well as the wild type and the per mutant males using the Affymetrix microarray. Two fly strains, a wild type Oregon R (OR) and a null mutant allele of per (per01) with the OR background for the second and third chromosomes, will be used. Virgin males and females will be collected without anesthesia within 6 hrs after eclosion and maintained individually in vials until experiments. Mated wild type females will be prepared by crossing to the wild type males the night before they are used for conditioning males. All flies will be raised at 25oC in a 12 h light/12 h dark cycle and the following behavioral experiments will be carried out during daytime. For courtship conditioning, a 3 day-old virgin male (either OR or per01) will be placed with a mated OR female in a conditioning chamber (15 mm x 5 mm in depth) containing fly food. As a control, a virgin male will be introduced into another conditioning chamber without a mated female. Seven hours after the male is introduced into the chamber, he will be anesthetized under CO2 gas or transferred to a new vial. For the anesthetized male, the head will be immediately excised and frozen with liquid nitrogen. The male fly transferred to a new vial will be decapitated 24 hrs after the end of the 7-hr training. Approximately thirty fly heads will be pooled for each of the 8-genotype/treatment groups, i.e., Oregon R 0-hr and 24-hr after conditioning, per01 0-hr and 24-hr after conditioning, in addition to the corresponding four naÃ¯ve controls. Total RNA will be extracted from the pooled heads using the TRIzol Reagent (Life Technologies) followed by an RNeasy (Qiagen) cleanup step and a DNAase I digestion step. After purification, the RNA will be resuspended in DEPC water and the absorbance will be checked at 260 and 280 nm for determination of sample purity and concentration. The GeneChip Drosophila Genome 2.0 Arrays will be used to quantify the levels of transcripts in each sample.

Project description:Disruption of learning and memory can occur for a number of reasons including chronic alcohol abuse, head injury, anoxia, and various neurodegenerative disorders such as Alzheimer’s disease. An understanding of the basic mechanisms underlying learning and memory is essential for prevention and treatment of such brain disorders. Recent studies unequivocally demonstrated the astonishing similarities in the basic mechanisms of learning and memory between divergent species, indicating that studies of experimentally tractable model organisms with relatively simpler nervous systems are likely to produce valuable information that will facilitate our understanding of learning and memory in higher organisms. We are studying the experience-dependent modification in Drosophila male courtship as a physiologically relevant model of learning and memory. The genome-scale gene-profiling analysis in this model system should provide us the basic information about learning-induced changes in gene expression, which will be the foundation of our future studies to identify genes and genetic pathways specifically involved in the formation of long-term memory. The proposed gene-profiling experiment is designed to identify candidate genes and genetic pathways involved in the formation of long-term memory in the Drosophila courtship conditioning. The specific aims of this project are to: 1) compare gene expression profiles between the naïve and trained wild type males using the GeneChip Drosophila Genome 2.0 Arrays and identify genes that are up- or down-regulated in the fly head immediately or 24-hr after the male fly is exposed to the condition where the long-term courtship memory is established (i.e., paired with a mated female for 7 hrs), 2) carry out the same analysis for the per null mutant males, 3) compare the identified genes between the 0 and 24-hr after training as well as the wild type and per mutant males, 4) identify candidate genes which might be involved in the long-term memory formation either upstream or downstream of per, 5) classify the candidate genes according to their putative cellular function and discern the possible genetic pathways necessary for the long-term memory formation. When a male fly is presented with a previously mated female, he will initially court her vigorously but his courtship activity is reduced over time. Subsequently, when paired with a virgin female, the male will display a reduced amount of courtship compared with naïve males with no prior courtship experience. This modification of courtship is called “courtship conditioning”. We found that this type of courtship memory lasts at least 5 days when a male is trained with a mated female for 7 hrs. We have also found that the period gene (per) that is involved in circadian rhythm regulation plays a critical role in the formation of long-term, but not short-term, courtship memory. Our hypotheses are 1) the long-lasting memory in 7 h-trained males is accompanied with characteristic temporal changes in gene expression in the brain, 2) per mutant males are defective in regulation of critical genes involved in long-term courtship memory, 3) the memory–related changes in gene expression can be detected by comparing total head transcript levels between trained and naïve as well as the wild type and the per mutant males using the Affymetrix microarray. Two fly strains, a wild type Oregon R (OR) and a null mutant allele of per (per01) with the OR background for the second and third chromosomes, will be used. Virgin males and females will be collected without anesthesia within 6 hrs after eclosion and maintained individually in vials until experiments. Mated wild type females will be prepared by crossing to the wild type males the night before they are used for conditioning males. All flies will be raised at 25oC in a 12 h light/12 h dark cycle and the following behavioral experiments will be carried out during daytime. For courtship conditioning, a 3 day-old virgin male (either OR or per01) will be placed with a mated OR female in a conditioning chamber (15 mm x 5 mm in depth) containing fly food. As a control, a virgin male will be introduced into another conditioning chamber without a mated female. Seven hours after the male is introduced into the chamber, he will be anesthetized under CO2 gas or transferred to a new vial. For the anesthetized male, the head will be immediately excised and frozen with liquid nitrogen. The male fly transferred to a new vial will be decapitated 24 hrs after the end of the 7-hr training. Approximately thirty fly heads will be pooled for each of the 8-genotype/treatment groups, i.e., Oregon R 0-hr and 24-hr after conditioning, per01 0-hr and 24-hr after conditioning, in addition to the corresponding four naïve controls. Total RNA will be extracted from the pooled heads using the TRIzol Reagent (Life Technologies) followed by an RNeasy (Qiagen) cleanup step and a DNAase I digestion step. After purification, the RNA will be resuspended in DEPC water and the absorbance will be checked at 260 and 280 nm for determination of sample purity and concentration. The GeneChip Drosophila Genome 2.0 Arrays will be used to quantify the levels of transcripts in each sample. Keywords: other

Dataset Information

Transcription profiling of Drosophila model of mitochondrial disease

Publications

Gene expression in a Drosophila model of mitochondrial disease.

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