Project description:The determination of glial fate in the developing embryonic nervous system of Drosophila is dependent on the master regulatory gene glial cells missing (gcm). gcm encodes a transcription factor and serves as a binary switch by inducing the glial fate and repressing the neuronal pathway. The ectopic expression of gcm throughout the CNS leads to excessive glia on the cost of presumptive neurons, whereas gcm loss-of-function embryos lack nearly all lateral glial cells. To date only little is known about the genetic program underlying glial differentiation. In order to identify glial specific genes downstream of gcm we performed a whole-genome microarray approach, where we compared the gcm gain-of-function situation (GOF) and, for the first time, the gcm loss-of-function (LOF) against wildtype in a time course experiment throughout embryogenesis. Intense filtering of differentially regulated genes on behalf of statistics as well as developmental means such as profile of differential regulation enabled us to identify more than 40 novel glial genes. The confirmation of these genes by in situ hybridization revealed temporal expression profiles in all or subsets of glial cells. We give a detailed description of the expression patterns of the candidate genes and adress their regulation in the glial pathway in different glial mutant backgrounds. Additionally we started to analyze mutant phenotypes of candidate genes to clarify their functional relevance for glial differentiation. Keywords: Drosophila,gliogenesis,time course,loss-of-function,gain-of-function
Project description:In order to analyze the global changes in gene expression resulting from loss of Fra signaling, we performed a microarray experiment comparing Drosophila embryos containing a loss of function fra[3] mutation to age matched wildtype embryos. RNA extracted from fra[3] mutant vs. wildtype embryos were hybridized to the Affymetrix GeneChip Drosophila Genome 2.0 .
Project description:We report here the application of magnetic cell separation in microarray experiment to understand Drosophila gliogenesis during early embryogenesis. At stage 11, neuroectodermal cells were labeled by a fusion transmembrane protein, mCD8-GFP, in the wild-type embryos as wells as in the embryos where glide/gcm was overexpressed. Following the cell dissociation, neuroectodermal cells were purified by using the commercial available anti-mCD8 microbeads. Microarray experiments based on prepared neuroectodermal cells from wild-type embryos verse glide/gcm overexpressed embryos at stage 11 have revealed 76 gene differentially regulated by overexpression of glide/gcm. Among them, there are 11 genes have already been shown to be regulated by glide/gcm in the previous publications. Additionally, we have identified 4 more new glide/gcm-regulated genes by in situ hybridization or immunohistochemical staining. It is very convincing that reduction of the tissue complexity by cell purification leads to low false positives in microarray experiment. Keywords: repeat sample
Project description:We report here the application of magnetic cell separation in microarray experiment to understand Drosophila gliogenesis during early embryogenesis. At stage 11, neuroectodermal cells were labeled by a fusion transmembrane protein, mCD8-GFP, in the wild-type embryos as wells as in the embryos where glide/gcm was overexpressed. Following the cell dissociation, neuroectodermal cells were purified by using the commercial available anti-mCD8 microbeads. Microarray experiments based on prepared neuroectodermal cells from wild-type embryos verse glide/gcm overexpressed embryos at stage 11 have revealed 76 gene differentially regulated by overexpression of glide/gcm. Among them, there are 11 genes have already been shown to be regulated by glide/gcm in the previous publications. Additionally, we have identified 4 more new glide/gcm-regulated genes by in situ hybridization or immunohistochemical staining. It is very convincing that reduction of the tissue complexity by cell purification leads to low false positives in microarray experiment. Keywords: other
Project description:We report here the application of magnetic cell separation in microarray experiment to understand Drosophila gliogenesis during early embryogenesis. At stage 11, neuroectodermal cells were labeled by a fusion transmembrane protein, mCD8-GFP, in the wild-type embryos as wells as in the embryos where glide/gcm was overexpressed. Following the cell dissociation, neuroectodermal cells were purified by using the commercial available anti-mCD8 microbeads. Microarray experiments based on prepared neuroectodermal cells from wild-type embryos verse glide/gcm overexpressed embryos at stage 11 have revealed 76 gene differentially regulated by overexpression of glide/gcm. Among them, there are 11 genes have already been shown to be regulated by glide/gcm in the previous publications. Additionally, we have identified 4 more new glide/gcm-regulated genes by in situ hybridization or immunohistochemical staining. It is very convincing that reduction of the tissue complexity by cell purification leads to low false positives in microarray experiment. Keywords: repeat sample
Project description:In order to analyze the global changes in gene expression resulting from loss of Fra signaling, we performed a microarray experiment comparing Drosophila embryos containing a loss of function fra[3] mutation to age matched wildtype embryos. RNA extracted from fra[3] mutant vs. wildtype embryos were hybridized to the Affymetrix GeneChip Drosophila Genome 2.0 . We selected Drosophila embryos at stage 13, which marks the onset of Fra expression in the embryonic CNS and the early stages of commissural axon guidance in the ventral nerve cord. RNA was extracted from both fra[3] and wildtype embryos. Hybridization experiments were performed on Affymetrix Drosophila Genome 2.0 microarrays.
Project description:We report here the application of magnetic cell separation in microarray experiment to understand Drosophila gliogenesis during early embryogenesis. At stage 11, neuroectodermal cells were labeled by a fusion transmembrane protein, mCD8-GFP, in the wild-type embryos as well as in the embryos where glide/gcm was overexpressed. Following the cell dissociation, neuroectodermal cells were purified by using the commercial available anti-mCD8 microbeads. Microarray experiments based on prepared neuroectodermal cells from wild-type embryos verse glide/gcm overexpressed embryos at stage 11 have revealed 76 gene differentially regulated by overexpression of glide/gcm.
Project description:To identify genes differentially expressed in the fatbody of Drosphila melanogaster bigmax mutants, a loss-of-function allele was generated by P-element mobilization. Mutant and wildtype first instar larvae were raised on two different sources of food, control and high-sugar media. When the animals reached the wandering third instar stage, animals were sacrificed and their fat bodies dissected. Total RNA was extracted, labeled fluorescently and hybridized competitively to Agilent's 4x44K Drosophila Gene Expression Microarrays. On each array, three different samples were analyzed: 1. wildtype animals raised on control food, 2. wildtype animals raised on high-sugar food and 3. bigmax mutant animals raised on high-sugar food.
Project description:In Saccharomyces cerevisiae, ubiquitylation of histone H2B signals methylation of histone H3 at lysine residues 4 (K4) and 79. These modifications occur at active genes but are believed to stabilize silent chromatin by limiting movement of silencing proteins away from heterochromatin domains. In the course of studying atypical phenotypes associated with loss of H2B ubiquitylation/H3K4 methylation, we discovered that these modifications are also required for cell wall integrity at high temperatures. We identified the silencing protein Sir4 as a dosage-suppressor of loss of H2B ubiquitylation, and showed that elevated Sir4 expression suppresses cell wall integrity defects by inhibiting the function of the Sir silencing complex. Using comparative transcriptome analysis, we identified a set of euchromatic genes - enriched in those required for cellular response to heat - whose expression is attenuated by loss of H2B ubiquitylation, but restored by disruption of Sir protein function. Finally, using DNA adenine methyltransferase identification, we found that Sir3 and Sir4 associate with genes that are silenced in the absence of H3K4 methylation. Our data reveal that H2B ubiquitylation/H3K4 methylation play an important role in limiting ectopic association of silencing proteins with euchromatic genes important for cell wall integrity and the response to heat. The transcriptome of four strains, wildtype, htbK123R, sir4delta,and htbK123R sir4delta, were sequenced using Illumina sequencing technology