Montserrat Corominas

The DrosDel Deletion Collection: A Drosophila Genomewide Chromosomal Deficiency Resource

We describe a second-generation deficiency kit for Drosophila melanogaster composed of molecularly mapped deletions on an isogenic background, covering ∼77% of the Release 5.1 genome. Using a previously reported collection of FRT-bearing P-element insertions, we have generated 655 new deletions and verified a set of 209 deletion-bearing fly stocks. In addition to deletions, we demonstrate how the P elements may also be used to generate a set of custom inversions and duplications, particularly useful for balancing difficult regions of the genome carrying haplo-insufficient loci. We describe a simple computational resource that facilitates selection of appropriate elements for generating custom deletions. Finally, we provide a computational resource that facilitates selection of other mapped FRT-bearing elements that, when combined with the DrosDel collection, can theoretically generate over half a million precisely mapped deletions.

Cell death-induced regeneration in wing imaginal discs requires JNK signalling

Regeneration and tissue repair allow damaged or lost body parts to be replaced. After injury or fragmentation of Drosophila imaginal discs, regeneration leads to the development of normal adult structures. This process is likely to involve a combination of cell rearrangement and compensatory proliferation. However, the detailed mechanisms underlying these processes are poorly understood. We have established a system to allow temporally restricted induction of cell death in situ. Using Gal4/Gal80 and UAS-rpr constructs, targeted ablation of a region of the disc could be performed and regeneration monitored without the requirement for microsurgical manipulation. Using a ptc-Gal4 construct to drive rpr expression in the wing disc resulted in a stripe of dead cells in the anterior compartment flanking the anteroposterior boundary, whereas a sal-Gal4 driver generated a dead domain that includes both anterior and posterior cells. Under these conditions, regenerated tissues were derived from the damaged compartment, suggesting that compartment restrictions are preserved during regeneration. Our studies reveal that during regeneration the live cells bordering the domain in which cell death was induced first display cytoskeletal reorganisation and apical-to-basal closure of the epithelium. Then, proliferation begins locally in the vicinity of the wound and later more extensively in the affected compartment. Finally, we show that regeneration of genetically ablated tissue requires JNK activity. During cell death-induced regeneration, the JNK pathway is activated at the leading edges of healing tissue and not in the apoptotic cells, and is required for the regulation of healing and regenerative growth.

In silico identification of novel selenoproteins in the Drosophila melanogaster genome

In selenoproteins, incorporation of the amino acid selenocysteine is specified by the UGA codon, usually a stop signal. The alternative decoding of UGA is conferred by an mRNA structure, the SECIS element, located in the 3′‐untranslated region of the selenoprotein mRNA. Because of the non‐standard use of the UGA codon, current computational gene prediction methods are unable to identify selenoproteins in the sequence of the eukaryotic genomes. Here we describe a method to predict selenoproteins in genomic sequences, which relies on the prediction of SECIS elements in coordination with the prediction of genes in which the strong codon bias characteristic of protein coding regions extends beyond a TGA codon interrupting the open reading frame. We applied the method to the Drosophila melanogaster genome, and predicted four potential selenoprotein genes. One of them belongs to a known family of selenoproteins, and we have tested experimentally two other predictions with positive results. Finally, we have characterized the expression pattern of these two novel selenoprotein genes.

ROS-Induced JNK and p38 Signaling Is Required for Unpaired Cytokine Activation during Drosophila Regeneration.

Upon apoptotic stimuli, epithelial cells compensate the gaps left by dead cells by activating proliferation. This has led to the proposal that dying cells signal to surrounding living cells to maintain homeostasis. Although the nature of these signals is not clear, reactive oxygen species (ROS) could act as a signaling mechanism as they can trigger pro-inflammatory responses to protect epithelia from environmental insults. Whether ROS emerge from dead cells and what is the genetic response triggered by ROS is pivotal to understand regeneration of Drosophila imaginal discs. We genetically induced cell death in wing imaginal discs, monitored the production of ROS and analyzed the signals required for repair. We found that cell death generates a burst of ROS that propagate to the nearby surviving cells. Propagated ROS activate p38 and induce tolerable levels of JNK. The activation of JNK and p38 results in the expression of the cytokines Unpaired (Upd), which triggers the JAK/STAT signaling pathway required for regeneration. Our findings demonstrate that this ROS/JNK/p38/Upd stress responsive module restores tissue homeostasis. This module is not only activated after cell death induction but also after physical damage and reveals one of the earliest responses for imaginal disc regeneration.

Screening of larval/pupal P-element induced lethals on the second chromosome in Drosophila melanogaster: clonal analysis and morphology of imaginal discs

We have carried out screens for lethal mutations on the second chromosome of Drosophila melanogaster that are associated with abnormal imaginal disc morphologies, particularly in the wing disc. From a collection of 164 P element-induced mutations with a late larva/pupa lethal phase we have identified 56 new loci whose gene products are required for normal wing disc development and for normal morphology of other larval organs. Genetic mosaics of these 56 mutant lines show clonal mutant phenotypes for 23 cell-viable mutations. These phenotypes result from altered cell parameters. Causal relationships between disc and clonal phenotypes are discussed.

Gene expression following induction of regeneration in Drosophila wing imaginal discs. Expression profile of regenerating wing discs

BackgroundRegeneration is the ability of an organism to rebuild a body part that has been damaged or amputated, and can be studied at the molecular level using model organisms. Drosophila imaginal discs, which are the larval primordia of adult cuticular structures, are capable of undergoing regenerative growth after transplantation and in vivo culture into the adult abdomen.ResultsUsing expression profile analyses, we studied the regenerative behaviour of wing discs at 0, 24 and 72 hours after fragmentation and implantation into adult females. Based on expression level, we generated a catalogue of genes with putative role in wing disc regeneration, identifying four classes: 1) genes with differential expression within the first 24 hours; 2) genes with differential expression between 24 and 72 hours; 3) genes that changed significantly in expression levels between the two time periods; 4) genes with a sustained increase or decrease in their expression levels throughout regeneration. Among these genes, we identified members of the JNK and Notch signalling pathways and chromatin regulators. Through computational analysis, we recognized putative binding sites for transcription factors downstream of these pathways that are conserved in multiple Drosophilids, indicating a potential relationship between members of the different gene classes. Experimental data from genetic mutants provide evidence of a requirement of selected genes in wing disc regeneration.ConclusionsWe have been able to distinguish various classes of genes involved in early and late steps of the regeneration process. Our data suggests the integration of signalling pathways in the promoters of regulated genes.

Imaginal discs: Renaissance of a model for regenerative biology.

Many animals display a capacity to regenerate tissues or even a complete body. One of the main goals of regenerative biology is to identify the genes and genetic networks necessary for this process. Drosophila offers an ideal model system for such studies. The wide range of genetic and genomic approaches available for use in flies has helped in initiating the deciphering of the mechanisms underlying regeneration, and the results may be applicable to other organisms, including mammals. Moreover, most models of regeneration require experimental manipulation, whereas in Drosophila discrete domains can be ablated by genetically induced methods. Here, we present a summary of current research into imaginal disc regeneration and discuss the power of this tissue as a tool for understanding the genetics of regeneration.

Functional dissection of the ash2 and ash1 transcriptomes provides insights into the transcriptional basis of wing phenotypes and reveals conserved protein interactions

BackgroundThe trithorax group (trxG) genes absent, small or homeotic discs 1 (ash1) and 2 (ash2) were isolated in a screen for mutants with abnormal imaginal discs. Mutations in either gene cause homeotic transformations but Hox genes are not their only targets. Although analysis of double mutants revealed that ash2 and ash1 mutations enhance each others phenotypes, suggesting they are functionally related, it was shown that these proteins are subunits of distinct complexes.ResultsThe analysis of wing imaginal disc transcriptomes from ash2 and ash1 mutants showed that they are highly similar. Functional annotation of regulated genes using Gene Ontology allowed identification of severely affected groups of genes that could be correlated to the wing phenotypes observed. Comparison of the differentially expressed genes with those from other genome-wide analyses revealed similarities between ASH2 and Sin3A, suggesting a putative functional relationship. Coimmunoprecipitation studies and immunolocalization on polytene chromosomes demonstrated that ASH2 and Sin3A interact with HCF (host-cell factor). The results of nucleosome western blots and clonal analysis indicated that ASH2 is necessary for trimethylation of the Lys4 on histone 3 (H3K4).ConclusionThe similarity between the transcriptomes of ash2 and ash1 mutants supports a model in which the two genes act together to maintain stable states of transcription. Like in humans, both ASH2 and Sin3A bind HCF. Finally, the reduction of H3K4 trimethylation in ash2 mutants is the first evidence in Drosophila regarding the molecular function of this trxG gene.

dKDM5/LID regulates H3K4me3 dynamics at the transcription-start site (TSS) of actively transcribed developmental genes

H3K4me3 is a histone modification that accumulates at the transcription-start site (TSS) of active genes and is known to be important for transcription activation. The way in which H3K4me3 is regulated at TSS and the actual molecular basis of its contribution to transcription remain largely unanswered. To address these questions, we have analyzed the contribution of dKDM5/LID, the main H3K4me3 demethylase in Drosophila, to the regulation of the pattern of H3K4me3. ChIP-seq results show that, at developmental genes, dKDM5/LID localizes at TSS and regulates H3K4me3. dKDM5/LID target genes are highly transcribed and enriched in active RNApol II and H3K36me3, suggesting a positive contribution to transcription. Expression-profiling show that, though weakly, dKDM5/LID target genes are significantly downregulated upon dKDM5/LID depletion. Furthermore, dKDM5/LID depletion results in decreased RNApol II occupancy, particularly by the promoter-proximal Pol lloser5 form. Our results also show that ASH2, an evolutionarily conserved factor that locates at TSS and is required for H3K4me3, binds and positively regulates dKDM5/LID target genes. However, dKDM5/LID and ASH2 do not bind simultaneously and recognize different chromatin states, enriched in H3K4me3 and not, respectively. These results indicate that, at developmental genes, dKDM5/LID and ASH2 coordinately regulate H3K4me3 at TSS and that this dynamic regulation contributes to transcription.

Genome-wide chromatin occupancy analysis reveals a role for ASH2 in transcriptional pausing

An important mechanism for gene regulation involves chromatin changes via histone modification. One such modification is histone H3 lysine 4 trimethylation (H3K4me3), which requires histone methyltranferase complexes (HMT) containing the trithorax-group (trxG) protein ASH2. Mutations in ash2 cause a variety of pattern formation defects in the Drosophila wing. We have identified genome-wide binding of ASH2 in wing imaginal discs using chromatin immunoprecipitation combined with sequencing (ChIP-Seq). Our results show that genes with functions in development and transcriptional regulation are activated by ASH2 via H3K4 trimethylation in nearby nucleosomes. We have characterized the occupancy of phosphorylated forms of RNA Polymerase II and histone marks associated with activation and repression of transcription. ASH2 occupancy correlates with phosphorylated forms of RNA Polymerase II and histone activating marks in expressed genes. Additionally, RNA Polymerase II phosphorylation on serine 5 and H3K4me3 are reduced in ash2 mutants in comparison to wild-type flies. Finally, we have identified specific motifs associated with ASH2 binding in genes that are differentially expressed in ash2 mutants. Our data suggest that recruitment of the ASH2-containing HMT complexes is context specific and points to a function of ASH2 and H3K4me3 in transcriptional pausing control.