Poly(A) mRNA-seq from yw and Twi-mCherry-LEXY(V416L) embryos subjected to different blue light exposure schemes
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ABSTRACT: Profiling of genes expression changes caused by the conditional perturbation of Twist function during different developmental time windows of Drosophila embryogenesis Twist was depleted from the nucleus continuously or post-gastrulation using the improved blue light-inducible mislocalisation system LEXY(slow). Gene expression changes between dark- and blue light-incubated embryos were profiled 2-4 h and 4-6 h after egg lay. yw embryos serve as a wild-type control. Poly(A) mRNA-seq was performed from three biological replicates for Twi-mCherry-LEXY(V416L) embryos and two biological replicates for yw embryos.
Project description:Drosophila midline(mid), encodes a T-box transcription factor that is required for cell fate specification and tissue morphogenesis during a series of developmental processes. However, little is known about the target of Mid. To identify the Mid regulated genes, we compared expression profiles of mRNA purified from wild-type and mid mutant embryos. Summary file is yw-mid.CHP. yw and mid mutant embryos were selected at later stage of development for RNA extraction and hybridization on Affymetrix microarrays. We identified mid mutant embryos using CyOact-GFP balancer chromosome.
Project description:We used FAIRE-seq to perform genome-wide profiling of open chromatin in 2-3 hour Drosophila embryos lacking maternal ZLD (zldM-) and in paired control embryos (yw). We demonstrate that ZLD is required to establish or maintain specific regions of open chromatin. Using single embryo RNA-seq data (from stage 5 yw and zldM- embryos) we show that loci that lose accessibility in zldM- embryos require ZLD for robust expression of associated genes. By comparing our FAIRE peaks to publicly available ChIP data (Xu et al. 2014) we demonstrate that ZLD is required for binding of the transcription factor Bicoid. To our surprise, not all ZLD-bound loci are less accessible in the mutant, suggesting that these regions rely on additional factors for open chromatin. These constitutively accessible ZLD-bound sites are enriched for the GAGA factor (GAF) binding motif, and we therefore propose that GAF may provide this additional function. We conclude that ZLD functions like a pioneer factor to define the cis-regulatory regions that drive gene expression during the MZT. Open chromatin profiling of 2-3 hour Drosophila embryos: embryos lacking maternal ZLD (zldM-, 3 replicates) and paired control embryos (yw, 2 replicates); Genomic DNA Inputs. Sequencing performed on Illumina HiSeq 2000.
Project description:In all animals, the initial events of embryogenesis are controlled by maternal gene products that are deposited into the developing oocyte. At some point after fertilization, control of embryogenesis is transferred to the zygotic genome in a process called the maternal to zygotic transition (MZT). During this time maternal RNAs are degraded and zygotic RNAs are transcribed1. A long standing question has been, what factors regulate these events? The recent findings that microRNAs and Smaugs mediate maternal transcript degradation brought new life to this old problem2,3, however, the transcription factors that activate zygotic gene expression remained elusive. A clue came from the finding that many early zygotic genes in Drosophila share a cis-regulatory heptamer motif, CAGGTAG and related sequences, collectively referred to as TAG-team sites4,5. We asked whether there was a dedicated transcription factor that interacts with these sites to activate early genes. Here we report the discovery of a zinc-finger protein, Zelda (Zld) that binds specifically to TAG-team sites, and is capable of activating transcription in transient transfection assays. Mutant embryos lacking zld are defective in the cellularization process, and fail to activate the transcription of many early zygotic genes involved in cellularization, sex determination, and dorsoventral patterning. Global expression profiling confirmed that Zld plays a key role in the activation of the early zygotic genome, and suggests that Zld may also play a role in maternal RNA degradation during the MZT since many RNAs are up-regulated in the absence of Zld. Experiment Overall Design: Total RNA samples were extracted from three replicate collections of 1-2 hr yw and M- zld embryos by TRIzol (invitrogen). A portion of the collected embryos was fixed and stained with DAPI; 90% were in nuclear cycles 8 to13. cDNA was prepared using the GeneChip® HT One-Cycle cDNA Synthesis Kit (Manufactured by Invitrogen for Affymetrix) and labeled with the BioArray⢠HighYield⢠RNA Transcript Labeling Kit (Enzo). Labeled probes were hybridized to Drosophila Genome 2 Affymetrix arrays and processed by a GeneChip Fluidics Station 400. Data were acquired and normalized by the GeneChip® Scanner 3000 and processed by the Affymetrix GeneChip Operating Software (GCOS). t-test analysis was performed on the data from three biological replicates.
Project description:Drosophila tdf, another name apontic (apt), encodes a bZIP transcription factor that is required for the development of trachea, heart, head and neural system. However, little is known about the target of TDF/Apt. To identify theTDF/ Apt regulated genes, we compared expression profiles of mRNA purified from wild-type and tdf mutant embryos. Summary file is emtdf-yw.CHP. yw and tdf mutant embryos were selected at later stage of development for RNA extraction and hybridization on Affymetrix microarrays. We identified tdf mutant embryos using CyOact-GFP balancer chromosome.
Project description:Multiprotein bridging factor 1(MBF1), a transcriptional co-activator that can regulate transcription by forming a bridge between the TATA-box-binding protein and gene-specific transcription factors. MBF1 is required for organ development and environmental stress responses. However, the targets of MBF1 is unknown. To identify the MBF1 regulated genes, we compared expression profiles of mRNA purified from wild-type and mbf1 mutant embryos. yw and mbf1 mutant embryos were selected at later stage of development for RNA extraction and hybridization on Affymetrix microarrays.
Project description:We performed ChIP-chip analysis using sheared chromatin isolated from Drosophila embryos of 1-3 hours in age. Keywords: high-resolution tiling array, drosophila, twist, dorsal, snail, weckle ChIP-chip of twist, dorsal, snail, and weckle
Project description:The conserved Mef2 transcription factor is a major regulator of gene expression and differentiation. Recent genomic studies have identified a large number of mef2-regulated target genes with distinct temporal expression profiles during Drosophila myogenesis. However, the question remains as to how a single transcription factor can control such diverse patterns of gene expression. The aim of this project was to investigate whether there are genes with different mef2-requirements for their expression during muscle differentiation in vivo during the development of Drosophila melanogaster. Experiment Overall Design: We used microarrays in conjunction with a mef2 allelic series to determine the gene expression profile in developing embryos at five different levels of mef2 activity. The allelic series extends from a null mutation, mef222.21, through to the control, via three hypomorphic alleles, mef2113, mef2424, and mef265. The series corresponds to different levels of Mef2 in the following order: control > 65 > 424 > 113 > 22.21. The control stock for the microarray analysis was dp cn a px sp, the stock used for the mutagenesis that produced mef265, mef2424 and mef2113. For the microarrays, 30 minute collections of control and mutant embryos for each mef2 allele were individually staged and pools of 150 embryos were then processed at mid stage 13. This corresponds to the early differentiation phase of muscle development and the expression of multiple muscle sarcomeric protein genes. Quadruplicate samples were assayed using Affymetrix® Genechips by the Flychip Drosophila microarray resource.
Project description:Embryos were collected, aged, mock-treated/treated with 40Gy gamma radiation, and allowed to recover for 1.5 hours. Targets from 3 sets of wild type (yw, w1118) and 2 sets of mutant (Dmp53NS) biological replicates were generated and the expression profiles were determined using Affymetrix Drosophila Genechip 1 arrays. Comparisons between the sample groups allow the identification of genes with radiation-responsive and Dmp53-dependent expression patterns. Experiment Overall Design: 3 sets of paired control and irradiated Wild Type and 2 sets of paired control and irradiated Mutant (Dmp53NS) biological replicates were analyzed
Project description:Expression profiling of D. melanogaster embryos expressing bagpipe ectopically in the mesoderm, measured in a timecourse of embryogenesis. Three one-hour timepoints were assayed (3.5-4.5, 4.5-5.5, 5.5-6.5 hrs after egg laying). Homozygous ectopically expressing embryos were collected using from a large scale cross of UAS-bin and twist-Gal4, 24B-Gal4 flies, in parallel to stage-matched controls. Four independent collections were performed at each timepoint. Total RNA was extracted and amplified. Ectopically expression and respective reference samples were hybridized together on INDAC oligo-arrays.
Project description:Expression profiling of D. melanogaster embryos expressing biniou ectopically in the mesoderm, measured in a timecourse of embryogenesis. Three one-hour timepoints were assayed (4-5, 5-6, 6-7 hrs after egg laying). Homozygous ectopically expressing embryos were collected using from a large scale cross of UAS-bin and twist-Gal4, 24B-Gal4 flies, in parallel to stage-matched controls. Four independent collections were performed at each timepoint. Total RNA was extracted and amplified. Ectopically expression and respective reference samples were hybridized together on INDAC oligo-arrays.