Developing tissues that contain mutant or compromised cells present risks to animal health. Accordingly, the appearance of a population of suboptimal cells in a tissue elicits cellular interactions that prevent their contribution to the adult. Here we report that this quality control process, cell competition, uses specific components of the evolutionarily ancient and conserved innate immune system to eliminate Drosophila cells perceived as unfit. We find that Toll-related receptors (TRRs) and the cytokine Spätzle (Spz) lead to NFκB-dependent apoptosis. Diverse “loser” cells require different TRRs and NFκB factors and activate distinct pro-death genes, implying that the particular response is stipulated by the competitive context. Our findings demonstrate a functional repurposing of components of TRRs and NFκB signaling modules in the surveillance of cell fitness during development.

SUMMARYOverexpression screens are used to explore gene functions in Drosophila, but this strategy suffers from the lack of comprehensive and systematic fly strain collections and efficient methods for generating such collections. Here, we present a strategy that could be used efficiently to generate large numbers of transgenic Drosophila strains, and a collection of 1149 UAS-ORF fly lines that were created with the site-specific ΦC31 integrase method. For this collection, we used a set of 655 genes that were cloned as two variants, either as an open reading frame (ORF) with a native stop codon or with a C-terminal 3xHA tag. To streamline the procedure for transgenic fly generation, we demonstrate the utility of injecting pools of plasmids into embryos, each plasmid containing a randomised sequence (barcode) that serves as a unique identifier for plasmids and, subsequently, fly strains. We also developed a swapping technique that facilitates the rapid exchange of promoters and epitope tags in vivo, expanding the versatility of the ORF collection. The work described here serves as the basis of a systematic library of Gal4/UAS-regulated transgenes.

The p53 tumor suppressor promotes apoptosis in response to DNA damage. Here we describe the Caenorhabditis elegans gene ced-13, which encodes a conserved BH3-only protein.We show that ced-13 mRNA accumulates following DNA damage, and that this accumulation is dependent on an intact C. elegans cep-1/p53 gene. We demonstrate that CED-13 protein physically interacts with the antiapoptotic Bcl-2-related protein CED-9. Furthermore, overexpression of ced-13 in somatic cells leads to the death of cells that normally survive, and this death requires the core apoptotic pathway of C. elegans. Recent studies have implicated two BH3-only proteins, Noxa and PUMA, in p53-induced apoptosis in mammals. Our studies suggest that in addition to the BH3-only protein EGL-1, CED-13 might also promote apoptosis in the C. elegans germ line in response to p53 activation. We propose that an evolutionarily conserved pathway exists in which p53 promotes cell death by inducing expression of two BH3-only genes.

To obtain insight into the role of the retinoblastoma susceptibility gene (Rb; also known as Rb1) in apoptosis, we analyzed Caenorhabditis elegans mutants lacking a functional lin-35 RB gene. We found that the loss of lin-35 function results in a decrease in constitutive germ cell apoptosis. We present evidence that lin-35 promotes germ cell apoptosis by repressing the expression of ced-9, an anti-apoptotic C. elegans gene that is orthologous to the human proto-oncogene BCL2. Furthermore, we show that the genes dpl-1 DP, efl-1 E2F and efl-2 E2F also promote constitutive germ cell apoptosis. However, in contrast to lin-35, dpl-1 (and probably also efl-1 and efl-2) promotes germ cell apoptosis by inducing the expression of the pro-apoptotic genes ced-4 and ced-3, which encode an APAF1-like adaptor protein and a pro-caspase, respectively. Based on these results, we propose that C. elegans orthologs of components of the RB tumor suppressor complex have distinct pro-apoptotic functions in the germ line and that the transcriptional regulation of components of the central apoptosis machinery is a critical determinant of constitutive germ cell apoptosis in C. elegans. Finally, we demonstrate that lin-35, dpl-1 and efl-2, but not efl-1, function either downstream of or in parallel to cep-1 p53 (also known as TP53) and egl-1 BH3-only to cause DNA damage-induced germ cell apoptosis. Our results have implications for the general mechanisms through which RB-like proteins control gene expression, the role of RB-, DP-and E2F-like proteins in apoptosis, and the regulation of apoptosis.

Transcription factors (TFs) are key regulators of cell fate. The estimated 755 genes that encode DNA binding domain-containing proteins comprise ∼5% of all Drosophila genes. However, the majority has remained uncharacterized so far due to the lack of proper genetic tools. We generated 594 site-directed transgenic Drosophila lines that contain integrations of individual UAS-TF constructs to facilitate spatiotemporally controlled misexpression in vivo. All transgenes were expressed in the developing wing, and two-thirds induced specific phenotypic defects. In vivo knockdown of the same genes yielded a phenotype for 50%, with both methods indicating a great potential for misexpression to characterize novel functions in wing growth, patterning, and development. Thus, our UAS-TF library provides an important addition to the genetic toolbox of Drosophila research, enabling the identification of several novel wing development-related TFs. In parallel, we established the chromatin landscape of wing imaginal discs by ChIP-seq analyses of five chromatin marks and RNA Pol II. Subsequent clustering revealed six distinct chromatin states, with two clusters showing enrichment for both active and repressive marks. TFs that carry such “bivalent” chromatin are highly enriched for causing misexpression phenotypes in the wing, and analysis of existing expression data shows that these TFs tend to be differentially expressed across the wing disc. Thus, bivalently marked chromatin can be used as a marker for spatially regulated TFs that are functionally relevant in a developing tissue.

Animal microRNAs (miRNA) are implicated in the control of nearly all cellular functions. Due to high sequence redundancy within the miRNA gene pool, loss of most of these 21-to 24-bp long RNAs individually does not cause a phenotype. Thus, only very few miRNAs have been associated with clear functional roles. We constructed a transgenic UAS-miRNA library in Drosophila melanogaster that contains 180 fly miRNAs. This library circumvents the redundancy issues by facilitating the controlled misexpression of individual miRNAs and is a useful tool to complement loss-of-function approaches. Demonstrating the effectiveness of our library, 78 miRNAs induced clear phenotypes. Most of these miRNAs were previously unstudied. Furthermore, we present a simple system to create GFP sensors to monitor miRNA expression and test direct functional interactions in vivo. Finally, we focus on the miR-92 family and identify a direct target gene that is responsible for the specific wing phenotype induced by the misexpression of miR-92 family members.

We created a site-directed UAS-ORF library of 655 growth-regulating genes in Drosophila. This library represents a large collection of genes regulating cell cycle, cell size, and proliferation and will be a valuable resource for studying growth regulation in vivo. By using misexpression of genes, we prevent problems arising from genetic redundancy and can uncover novel gene functions. To validate the usefulness of this library, we screened for Wingless (Wg) pathway components. We used a combination of experimental and bioinformatic approaches to predict candidates and identified three serine/threonine kinases as regulators of Wg signaling. We show that one of these, Nek2, optimizes pathway response by direct phosphorylation of Dishevelled. In addition, we describe functional relations for roughly 5% of all Drosophila genes and identify a large number of genes that regulate cell size, proliferation, and final organ size upon misexpression.