Unknown,Transcriptomics,Genomics,Proteomics

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Characterisation of the in-vivo miRNA landscape in Drosophila ribonuclease mutants reveals Pacman mediated regulation of the highly conserved let-7 cluster during apoptotic processes

ABSTRACT: RNA extracted from Drosophila wandering L3 wing imaginal discs was 2S rRNA depleted and prepared for miRNA sequencing using the QIAseq miRNA Library kit. The experiment was performed on Pacman and Dis3L2 null mutants and their respective isogenic control lines with four replicates of each condition.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Ben Towler

PROVIDER: E-MTAB-13781 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:An analysis of the transcriptional profiles of weak genetic perturbations generated via P-elements measured as heterozygotes in wing imaginal discs of Drosophila melanogaster. Abstract: A major objective of genomics is to elucidate the mapping between genotypic and phenotypic space as a step toward understanding how small changes in gene function can lead to elaborate phenotypic changes. One approach that has been utilized is to examine overall patterns of co-variation between phenotypic variables of interest, such as morphology, physiology and behavior, and underlying aspects of gene activity, in particular transcript abundance on a genome wide scale. Numerous studies have demonstrated that such patterns of co-variation occur, although these are often between samples with large numbers of unknown genetic differences (different strains, or even species) or perturbations of large effect (sexual dimorphism, or strong loss of function mutations), that may represent physiological changes outside of the normal experiences of the organism. We used weak mutational perturbations in genes affecting wing development in Drosophila melanogaster, that influence wing shape relative to a co-isogenic wild-type. We profiled transcription of 1150 genes expressed during wing development in 27 heterozygous mutants, as well as their co-isogenic wild type and one additional wild-type strain. Despite finding clear evidence of expression differences between mutants and wild-type, transcriptional profiles did not co-vary strongly with shape, suggesting that information from transcriptional profiling may not generally be predictive of final phenotype. We discuss these results in the light of possible attractor states of gene expression, and how this would affect interpretation of co-variation between transcriptional profiles and other phenotypes. Total RNA was extracted from pools of wing imaginal discs for each mutant strain as well as for their co-isogenic wild-type, and one additional wild-type strain. RNA was extracted using a modified protocol using the Qiagen RNAeasy kit.

Project description:Wound healing is an essential homeostatic mechanism that maintains the epithelial barrier integrity after tissue damage. Although we know the main events participating in the healing of a wound, many of the underlying molecular mechanisms remain unclear. Genetically amenable systems, such as wound healing in Drosophila imaginal discs, do not model all aspects of the repair process, but allow exploring many unanswered features of the healing response; e.g., which are the signal(s) responsible for initiating tissue remodeling? How is the sealing of the epithelia achieved? Or which are the inhibitory cues that cancel the healing machinery upon completion? Answering these and other questions demands in first place the identification and functional analysis of wound-specific genes. A variety of different microarray analyses of murine and humans have identified characteristic profiles of gene expression at the wound site, however, very few functional studies in healing regulation have been carried out. We developed an experimentally controlled method to culture imaginal discs that allows live imaging and biochemical analysis and is healing-permissive. Employing this approach, we performed a comparative genome-wide profiling between those Drosophila imaginal cells actively involved in healing versus their non-engaged siblings. This lets us identify a set of potential wound-specific genes. Importantly, besides identifying and categorizing new genes, we functionally tested many of their gene products by genetic interference and overexpression in a healing assay. This non-saturated analysis defines a relevant set of new genes whose changes in expression levels are functionally significant for proper tissue repair. There is promise that our newly identified wound-healing genes will guide future work in the more complex mammalian wound response. We developed a healing-permissive in vitro culture system for fly imaginal discs: we used one-channel microarrays for comparing healing-engaged cells (showing activation of the JNK signaling cascade) to cells not participating in healing (silent JNK activity) in wounded wing imaginal discs in culture. Employing this method, we aimed detecting the relevant genes involved in disc healing through microarray analysis. We compared cells actively involved in healing to those not involved and identified a whole set of upregulated or downregulated genes. They were annotated, clustered by expression profiles, chromosomal locations and presumptive functions. Most importantly, we functionally tested them genetically in a healing assay.

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Characterisation of the in-vivo miRNA landscape in Drosophila ribonuclease mutants reveals Pacman mediated regulation of the highly conserved let-7 cluster during apoptotic processes

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