Transcription profiling of staged Zebra fish embryos at 2 hour intervals over a 24 h period over period of segmentation, when the nervous system is forming
ABSTRACT: We isolated total RNA from staged embryos at 2 hour intervals over a 24 h period. Total RNA from 8 h embryos was used as a reference for the time course. This expression study used a >16,000 oligonucleotide (65-mers) probe set from Compugen to examine changes in gene expression in wild-type Danio rerio during development, emphasizing the period of segmentation, when the nervous system is forming. Embryos were provided by Scientific Hatcheries.
Project description:The effect of a year-long 10 reduction in water temperature on global gene expression in tail skeletal muscle from adult, male zebrafish was determined using a long oligonucleotide probe set (16,399 65mers from Compugen) spotted onto glass slides. Outbred male zebrafish were obtained from a commercial supplier (Liles Tropical Fish, FL) at 6 months of age. Fish were maintained at 28 until 10 months of age. (see Gerhard et al., Exp Gerontol 37,1055-68, 2002) For temperature reduction, water temperature was decreased by 2.5 per week for 4 weeks. Fish were maintained until 22 months of age. Fish were fed fish flakes (Wardely Corp. Secaucus, NJ) twice per day. Each feeding was a discrete event in which a small measured amount of granular food was sprinkled on the water surface. A second small aliquot was offered if the first has been eaten within a few minutes. The feeding stopped when food from the previous aliquot has not been eaten and feeding behavior has ceased. By providing a consecutive series of small aliquots until food is no longer accepted, every member of the tank has an opportunity to eat until satiety, yet the amount of uneaten food is minimized. At 22 months of age, fish were euthanized by decapitation. Total RNA was harvested from a pool of tail muscle samples from 5 fish. Two independent pools of 5 fish per pool were collected from fish maintained at 18 and 28. Flip-dye hybridizations were performed on each pool for a total of 4 hybridizations in this study. Experiments DAR011d0001 and DAR011d0002 are flip-dye hybridizations from Pool 1. Experiments DAR011d0003 and DAR011d004 are flip-dye hybridizations from Pool 2.
Project description:The strongest risk factor for developing Alzheimer's Disease (AD) is age. Here, we study the relationship between ageing and AD using a systems biology approach that employs a Drosophila (fruitfly) model of AD in which the flies overexpress the human Aβ42 peptide. We identified 712 genes that are differentially expressed between control and Aβ-expressing flies. We further divided these genes according to how they change over the animal's lifetime and discovered that the AD-related gene expression signature is age- independent. We have identified a number of differentially expressed pathways that are likely to play an important role in the disease, including oxidative stress and innate immunity. In particular, we uncovered two new modifiers of the Aβ phenotype, namely Sod3 and PGRP-SC1b. Transcript level measured using microarrays in biological quadruplicate (except day 20 which is in biological triplicate), each array is Aβ vs control at the same timepoint (defined by % survival), one replicate per array with dye-swaps.
Project description:Dichaete is a developmentally important transcription factor, known to be involved in basic biological processes including segmentation and nervous system development among others. The aim of this experiment was to gain further insight into the role of Dichaete during early embryogenesis, by looking at the disruption of gene expression in Dichaete mutants. Stage 10-11 embryos (5 and 7.5 hours after egg laying) from a cross between Dr72/TM3, twi-GAL4 UAS-Gfp Dr513/TM3, twi-GAL4 UAS-Gfp, were hand picked under a fluorescence dissecting microscope. GFP negative homozygous Dichaete mutant embryos and their heterozygous single GFP positive siblings were collected and approximately 150 embryos per sample were stored frozen in Trizol
Project description:Dichaete is a developmentally important transcription factor, known to be involved in basic biological processes including segmentation and nervous system development among others. The aim of this experiment was to gain further insight into the role of Dichaete during early embryogenesis, specifically looking at targets in the midline using dominant negative constructs expressed via the UAS/Gal4 system, using prosGal4 to drive expression in developing neuroblasts. 4 independent biological replicates. 3.5-4.5h old embryos were collected, and the RNA was extracted using Trizol. The UAS-dominant negative construct expressing embryos were compared to UAS-GFP expressing controls. Construct expression was driven using prosGal4.
Project description:Dichaete is a developmentally important transcription factor, known to be involved in basic biological processes including segmentation and nervous system development among others. The aim of this experiment was to gain further insight into the role of Dichaete during early embryogenesis, specifically looking at targets in the midline using dominant negative constructs expressed via the UAS/Gal4 system, using simGal4 to drive expression in the midline. 4 independent biological replicates. 3.5-4.5h old embryos were collected, and the RNA was extracted using Trizol. The UAS-dominant negative construct expressing embryos were compared to UAS-GFP expressing controls. Construct expression was driven using simgal4.
Project description:The LIM homeodomain transcription factor Lmx1a is a very potential inducer of stem cells towards dopaminergic neurons. Despite several studies on the function of this gene, the exact in vivo role of Lmx1a in mesodiencephalic dopamine (mdDA) neuronal specification is still not understood. To analyze the genes functioning downstream of Lmx1a, we performed expression microarray analysis of LMX1A overexpressing MN9D dopaminergic cells. Several interesting regulated genes were identified, based on their regulation in other, previously generated expression arrays, and their expression pattern in the developing mdDA neuronal field. Post analysis through in vivo expression analysis in Lmx1a mouse mutant (drJ/drJ) embryos demonstrated a clear decrease in expression of the genes Grb10 and Rgs4, in and adjacent to the rostral and dorsal mdDA neuronal field and within the Lmx1a expression domain. Interestingly, the DA marker Vmat2 was significantly up-regulated as a consequence of increased LMX1A dose, and subsequent analysis on Lmx1a mutant E14.5 and adult tissue revealed a significant decrease in Vmat2 expression in mdDA neurons. Taken together, microarray analysis of an LMX1A overexpression cell system resulted in the identification of novel downstream targets of Lmx1A in mdDA neurons: Grb10, Rgs4 and Vmat2. RNA was isolated from MN9D cells. Each experimental sample consisted of a RNA pool derived from 3 separate 10-cm dishes containing Lmx1a overexpressing MN9D cells (transfected with pcDNA3.1(-)-Lmx1a). microarray analysis was performed in triplicate, each experimental sample was hybridized to the same reference pool of RNA derived from 9 10-cm dishes containing control MN9D cells (transfected with empty pcDNA3.1(-)). On each of three microarray samples, dye swap was performed to correct for dye effects.
Project description:Responses to social cues, such as pheromones, can be modified by genotype, physiology, or environmental context. Honey bee queens produce a pheromone (queen mandibular pheromone; QMP) which regulates many aspects of worker bee behavior and physiology. Forager honey bees are less responsive to QMP than young nurse bees engaged in brood care, suggesting that physiological changes associated with behavioral maturation may modulate response to this pheromone. Since cGMP is a major regulator of behavioral maturation in honey bee workers, we examined its role in modulating worker responses to QMP. Treatment with a cGMP analog, 8-Br-cGMP, resulted in significant reductions in both behavioral and physiological responses to QMP in young caged workers. Treatment significantly reduced attraction to QMP (the retinue response) and inhibited the QMP-mediated increase in vitellogenin levels in the fat bodies of worker bees. Genome-wide analysis of brain gene expression patterns demonstrated that cGMP has a larger effect on expression levels than QMP, and that QMP has specific effects in the presence of cGMP, suggesting that some responses to QMP may be dependent on an individual bees physiological state. Several functional gene categories were significantly differentially expressed, including genes involved in regulating GTPase activity, phototransduction, immunity, and carboxylic acid transmembrane transporter activity. Overall, our data suggest that cGMP-mediated processes play a large role in modulating responses to queen pheromone in honey bees, at the behavioral, physiological and molecular levels.
Project description:Timecourse of gene expression changes in Drosophila SoxN homozygous mutant embryos compared with their heterozygous siblings, from stage 7 to 13 of embryonic development. Five time points: stage 7-8, stage 9, stage 10, stage 11 and stage 12-13. Four biological replicates per time point. Two conditions: SoxN homoxygous vs SoxN heterozygous mutant embryos.