RESUMEN
Germline genes often become re-expressed in soma-derived human cancers as "cancer/testis antigens" (CTAs), and piRNA (PIWI-interacting RNA) pathway proteins are found among CTAs. However, whether and how the piRNA pathway contributes to oncogenesis in human neoplasms remain poorly understood. We found that oncogenic Ras combined with loss of the Hippo tumor suppressor pathway reactivates a primary piRNA pathway in Drosophila somatic cells coincident with oncogenic transformation. In these cells, Piwi becomes loaded with piRNAs derived from annotated generative loci, which are normally restricted to either the germline or the somatic follicle cells. Negating the pathway leads to increases in the expression of a wide variety of transposons and also altered expression of some protein-coding genes. This correlates with a reduction in the proliferation of the transformed cells in culture, suggesting that, at least in this context, the piRNA pathway may play a functional role in cancer.
Asunto(s)
Transformación Celular Neoplásica/patología , Drosophila/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Línea Celular , Proliferación Celular , Transformación Celular Neoplásica/genética , Células Cultivadas , Elementos Transponibles de ADN/genética , Drosophila/citología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Regulación de la Expresión Génica , Silenciador del Gen , Ovario/citología , Transducción de Señal/genéticaRESUMEN
RNase P RNA (RPR), the catalytic subunit of the essential RNase P ribonucleoprotein, removes the 5' leader from precursor tRNAs. The ancestral eukaryotic RPR is a Pol III transcript generated with mature termini. In the branch of the arthropod lineage that led to the insects and crustaceans, however, a new allele arose in which RPR is embedded in an intron of a Pol II transcript and requires processing from intron sequences for maturation. We demonstrate here that the Drosophila intronic-RPR precursor is trimmed to the mature form by the ubiquitous nuclease Rat1/Xrn2 (5') and the RNA exosome (3'). Processing is regulated by a subset of RNase P proteins (Rpps) that protects the nascent RPR from degradation, the typical fate of excised introns. Our results indicate that the biogenesis of RPR in vivo entails interaction of Rpps with the nascent RNA to form the RNase P holoenzyme and suggests that a new pathway arose in arthropods by coopting ancient mechanisms common to processing of other noncoding RNAs.
Asunto(s)
Empalme Alternativo , Proteínas de Drosophila/genética , Subunidades de Proteína/genética , ARN Mensajero/genética , ARN de Transferencia/genética , Ribonucleasa P/genética , Animales , Evolución Biológica , Línea Celular , Biología Computacional/métodos , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Embrión no Mamífero , Exones , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Intrones , Macrófagos/citología , Macrófagos/enzimología , Masculino , Conformación de Ácido Nucleico , Subunidades de Proteína/metabolismo , Proteolisis , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Ribonucleasa P/metabolismoRESUMEN
Polycomb group (PcG) complexes PRC1 and PRC2 are well known for silencing specific developmental genes. PRC2 is a methyltransferase targeting histone H3K27 and producing H3K27me3, essential for stable silencing. Less well known but quantitatively much more important is the genome-wide role of PRC2 that dimethylates â¼70% of total H3K27. We show that H3K27me2 occurs in inverse proportion to transcriptional activity in most non-PcG target genes and intergenic regions and is governed by opposing roaming activities of PRC2 and complexes containing the H3K27 demethylase UTX. Surprisingly, loss of H3K27me2 results in global transcriptional derepression proportionally greatest in silent or weakly transcribed intergenic and genic regions and accompanied by an increase of H3K27ac and H3K4me1. H3K27me2 therefore sets a threshold that prevents random, unscheduled transcription all over the genome and even limits the activity of highly transcribed genes. PRC1-type complexes also have global roles. Unexpectedly, we find a pervasive distribution of histone H2A ubiquitylated at lysine 118 (H2AK118ub) outside of canonical PcG target regions, dependent on the RING/Sce subunit of PRC1-type complexes. We show, however, that H2AK118ub does not mediate the global PRC2 activity or the global repression and is predominantly produced by a new complex involving L(3)73Ah, a homolog of mammalian PCGF3.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Complejo Represivo Polycomb 2/metabolismo , Transcripción Genética , Animales , Drosophila melanogaster/metabolismo , Silenciador del Gen , Genoma , Histonas/metabolismo , MetilaciónRESUMEN
Ribonuclease P (RNase P) is an essential enzyme required for 5'-maturation of tRNA. While an RNA-free, protein-based form of RNase P exists in eukaryotes, the ribonucleoprotein (RNP) form is found in all domains of life. The catalytic component of the RNP is an RNA known as RNase P RNA (RPR). Eukaryotic RPR genes are typically transcribed by RNA polymerase III (pol III). Here we showed that the RPR gene in Drosophila, which is annotated in the intron of a pol II-transcribed protein-coding gene, lacks signals for transcription by pol III. Using reporter gene constructs that include the RPR-coding intron from Drosophila, we found that the intron contains all the sequences necessary for production of mature RPR but is dependent on the promoter of the recipient gene for expression. We also demonstrated that the intron-coded RPR copurifies with RNase P and is required for its activity. Analysis of RPR genes in various animal genomes revealed a striking divide in the animal kingdom that separates insects and crustaceans into a single group in which RPR genes lack signals for independent transcription and are embedded in different protein-coding genes. Our findings provide evidence for a genetic event that occurred approximately 500 million years ago in the arthropod lineage, which switched the control of the transcription of RPR from pol III to pol II.
Asunto(s)
Drosophila/genética , Evolución Molecular , ARN Catalítico/genética , Ribonucleasa P/genética , Animales , Crustáceos/genética , Drosophila melanogaster/genética , Regulación de la Expresión Génica , Genómica , Intrones/genética , Regiones Promotoras Genéticas , ARN Polimerasa III/genética , ARN de Transferencia/genéticaRESUMEN
The use of time series profiling to identify groups of functionally related genes (synexpression groups) is a powerful approach for the discovery of gene function. Here we apply this strategy during Ras(V12) immortalization of Drosophila embryonic cells, a phenomenon not well characterized. Using high-resolution transcriptional time-series datasets, we generated a gene network based on temporal expression profile similarities. This analysis revealed that common immortalized cells are related to adult muscle precursors (AMPs), a stem cell-like population contributing to adult muscles and sharing properties with vertebrate satellite cells. Remarkably, the immortalized cells retained the capacity for myogenic differentiation when treated with the steroid hormone ecdysone. Further, we validated in vivo the transcription factor CG9650, the ortholog of mammalian Bcl11a/b, as a regulator of AMP proliferation predicted by our analysis. Our study demonstrates the power of time series synexpression analysis to characterize Drosophila embryonic progenitor lines and identify stem/progenitor cell regulators.
Asunto(s)
Línea Celular Transformada , Drosophila/embriología , Células Madre/citología , Animales , Diferenciación Celular , Proliferación Celular , Embrión no Mamífero/citología , Regiones Promotoras Genéticas , Proteína de Retinoblastoma/metabolismo , Transcripción GenéticaRESUMEN
ErbB receptors, including the epidermal growth factor receptor (Egfr), are activated by EGF ligands to govern cell proliferation, survival, migration and differentiation. The different EGF-induced cell responses in development are regulated by deployment of multiple ligands. These inputs, however, engage only a limited number of intracellular pathways and are thought to elicit specific responses by regulating the amplitude or duration of the intracellular signal. The single Drosophila Egfr has four ligands: three of the TGF-α-type and a single neuregulin-like called vein (vn). Here, we used mutant combinations and gene replacement to determine the constraints of ligand specificity in development. Mutant analysis revealed extensive ligand redundancy in embryogenesis and wing development. Surprisingly, we found that the essential role of vn in development could be largely replaced by expression of any TGF-α ligand, including spitz (spi), in the endogenous vn pattern. vn mutants die as white undifferentiated pupae, but the rescued individuals showed global differentiation of adult body parts. Spi is more potent than Vn, and the best morphological rescue occurred when Spi expression was reduced to achieve an intracellular signaling level comparable to that produced by Vn. Our results show that the developmental repertoire of a strong ligand like Spi is flexible and at the appropriate level can emulate the activity of a weak ligand like Vn. These findings align with a model whereby cells respond similarly to an equivalent quantitative level of an intracellular signal generated by two distinct ligands regardless of ligand identity.
Asunto(s)
Drosophila/embriología , Drosophila/metabolismo , Neurregulinas/metabolismo , Factor de Crecimiento Transformador alfa/metabolismo , Alas de Animales/metabolismo , Animales , Proteínas de Drosophila/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Proteínas de la Membrana/metabolismo , Alas de Animales/embriologíaRESUMEN
The acquisition of flight contributed to the success of insects and winged forms are present in most orders. Key to understanding the origin of wings will be knowledge of the earliest postembryonic events promoting wing outgrowth. The Drosophila melanogaster wing is intensely studied as a model appendage, and yet little is known about the beginning of wing outgrowth. Vein (Vn) is a neuregulin-like ligand for the EGF receptor (Egfr), which is necessary for global development of the early Drosophila wing disc. vn is not expressed in the embryonic wing primordium and thus has to be induced de novo in the nascent larval wing disc. We find that Decapentaplegic (Dpp), a Bone Morphogenetic Protein (BMP) family member, provides the instructive signal for initiating vn expression. The signaling involves paracrine communication between two epithelia in the early disc. Once initiated, vn expression is amplified and maintained by autocrine signaling mediated by the E-twenty six (ETS)-factor PointedP2 (PntP2). This interplay of paracrine and autocrine signaling underlies the spatial and temporal pattern of induction of Vn/Egfr target genes and explains both body wall development and wing outgrowth. It is possible this gene regulatory network governing expression of an EGF ligand is conserved and reflects a common origin of insect wings.
Asunto(s)
Proteínas de Drosophila/metabolismo , Receptores ErbB/metabolismo , Regulación de la Expresión Génica/fisiología , Receptores de Péptidos de Invertebrados/metabolismo , Transducción de Señal/fisiología , Alas de Animales/crecimiento & desarrollo , Animales , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/genética , Drosophila melanogaster , Receptores ErbB/genética , Neurregulinas/biosíntesis , Neurregulinas/genética , Receptores de Péptidos de Invertebrados/genéticaRESUMEN
Mutations in Drosophila rumi result in a temperature-sensitive loss of Notch signaling. Rumi is a protein O-glucosyltransferase that adds glucose to EGF repeats with a C-X-S-X-P-C consensus sequence. Eighteen of the 36 EGF repeats in the Drosophila Notch receptor contain the consensus O-glucosylation motif. However, the contribution of individual O-glucose residues on Notch to the regulation of Notch signaling is not known. To address this issue, we carried out a mutational analysis of these glucosylation sites and determined their effects on Notch activity in vivo. Our results indicate that even though no single O-glucose mutation causes a significant decrease in Notch activity, all of the glucose residues on Notch contribute in additive and/or redundant fashions to maintain robust signaling, especially at higher temperatures. O-glucose motifs in and around the ligand-binding EGF repeats play a more important role than those in other EGF repeats of Notch. However, a single O-glucose mutation in EGF12 can be compensated by other O-glucose residues in neighboring EGF repeats. Moreover, timecourse cell aggregation experiments using a rumi null cell line indicate that a complete lack of Rumi does not affect Notch-Delta binding at high temperature. In addition, rumi fully suppresses the gain-of-function phenotype of a ligand-independent mutant form of Notch. Our data suggest that, at physiological levels of Notch, the combined effects of multiple O-glucose residues on this receptor allow productive S2 cleavage at high temperatures and thereby serve as a buffer against temperature-dependent loss of Notch signaling.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Glucosiltransferasas/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Genoma de los Insectos , Glucosa/metabolismo , Glucosiltransferasas/genética , Mutación , Receptores Notch/genética , Temperatura , TransgenesRESUMEN
Expression of activated Ras, RasV12, provides Drosophila cultured cells with a proliferation and survival advantage that simplifies the generation of continuous cell lines. Here, we used lineage-restricted RasV12 expression to generate continuous cell lines of muscle, glial, and epithelial cell type. Additionally, cell lines with neuronal and hemocyte characteristics were isolated by cloning from cell cultures established with broad RasV12 expression. Differentiation with the hormone ecdysone caused maturation of cells from mesoderm lines into active muscle tissue and enhanced dendritic features in neuronal-like lines. Transcriptome analysis showed expression of key cell-type-specific genes and the expected alignment with single-cell sequencing and in situ data. Overall, the technique has produced in vitro cell models with characteristics of glia, epithelium, muscle, nerve, and hemocyte. The cells and associated data are available from the Drosophila Genomic Resource Center.
Fruit flies are widely used in the life and biomedical sciences as models of animal biology. They are small in size and easy to care for in a laboratory, making them ideal for studying how the body works. There are, however, some experiments that are difficult to perform on whole flies and it would be advantageous to use populations of fruit fly cells grown in the laboratory known as cell cultures instead. Unlike studies in humans and other mammals, which for ethical and practical reasons heavily rely on cell cultures, few studies have used fruit fly cell cultures. Recent work has shown that having an always active version of a gene called Ras in fruit fly cells helps the cells to survive and grow in cultures, making it simpler to generate new fruit fly cell lines compared with traditional methods. However, the methods used to express activated Ras result in cell lines that can be a mixture of many different types of cell, which limits how useful they are for research. Here, Coleman-Gosser, Hu, Raghuvanshi, Stitzinger et al. aimed to use Ras to generate a collection of cell lines from specific types of fruit fly cells in the muscle, nervous system, blood and other parts of the body. The experiments show that selectively expressing activated Ras in an individual type of cell enables them to outcompete other cells in culture to generate a cell line consisting only of the cell type of interest. The new cell lines offer models for experiments that more closely reflect their counterparts in flies. For example, the team were able to recapitulate how fly muscles develop by treating one of the cell lines with a hormone called ecdysone, which triggered the cells to mature into active muscle cells that spontaneously contract and relax. In the future, the new cell lines could be used for various experiments including high throughput genetic screening or testing the effects of new drugs and other compounds. The method used in this work may also be used by other researchers to generate more fruit fly cell lines.
Asunto(s)
Drosophila , Hemocitos , Animales , Drosophila/genética , Neuroglía/metabolismo , Línea Celular , Músculos , Drosophila melanogaster/genéticaRESUMEN
Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high-throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. Here we found expression of the conserved oncogene Ras(V12) (a constitutively activated form of Ras) profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The lines have undergone more than 90 population doublings and therefore constitute continuous cell lines. Most lines are composed of spindle-shaped cells of mesodermal type. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts) tumor suppressor gene was targeted. We successfully created several cell lines and found that these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of Ras(V12) is a powerful genetic mechanism to promote proliferation in Drosophila primary culture cells and serves as an efficient means to generate continuous cell lines of a given genotype.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Línea Celular Transformada/fisiología , Drosophila/citología , Drosophila/genética , Animales , Línea Celular/clasificación , Proliferación Celular , Drosophila/embriología , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Expresión Génica , Genotipo , Cariotipificación , Masculino , Proteína Oncogénica p21(ras)/genética , Proteína Oncogénica p21(ras)/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
Differential gene expression is the major mechanism underlying the development of specific body regions. Here we assessed the role of genes differentially expressed in the Drosophila wing imaginal disc, which gives rise to two distinct adult structures: the body wall and the wing. Reverse genetics was used to test the function of uncharacterized genes first identified in a microarray screen as having high levels of expression in the presumptive wing. Such genes could participate in elaborating the specific morphological characteristics of the wing. The activity of the genes was modulated using misexpression and RNAi-mediated silencing. Misexpression of eight of nine genes tested caused phenotypes. Of 12 genes tested, 10 showed effective silencing with RNAi transgenes, but only 3 of these had resulting phenotypes. The wing phenotypes resulting from RNAi suggest that CG8780 is involved in patterning the veins in the proximal region of the wing blade and that CG17278 and CG30069 are required for adhesion of wing surfaces. Venation and apposition of the wing surfaces are processes specific to wing development providing a correlation between the expression and function of these genes. The results show that a combination of expression profiling and tissue-specific gene silencing has the potential to identify new genes involved in wing development and hence to contribute to our understanding of this process. However, there are both technical and biological limitations to this approach, including the efficacy of RNAi and the role that gene redundancy may play in masking phenotypes.
Asunto(s)
Proteínas de Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Alas de Animales/fisiología , Secuencia de Aminoácidos , Animales , Células Cultivadas , Cruzamientos Genéticos , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Femenino , Hibridación in Situ , Masculino , Datos de Secuencia Molecular , Fenotipo , Pupa/citología , Pupa/metabolismo , ARN Interferente Pequeño/farmacología , Homología de Secuencia de AminoácidoRESUMEN
A long-standing problem with analyzing transgene expression in tissue-culture cells is the variation caused by random integration of different copy numbers of transfected transgenes. In mammalian cells, single transgenes can be inserted by homologous recombination but this process is inefficient in Drosophila cells. To tackle this problem, our group, and the Cherbas group, used recombination-mediated cassette exchange (RMCE) to introduce single-copy transgenes into specific locations in the Drosophila genome. In both cases, ÏC31 was used to catalyze recombination between its target sequences attP in the genome, and attB flanking the donor sequence. We generated cell lines de novo with a single attP-flanked cassette for recombination, whereas, Cherbas et al. introduced a single attP-flanked cassette into existing cell lines. In both approaches, a 2-drug selection scheme was used to select for cells with a single copy of the donor sequence inserted by RMCE and against cells with random integration of multiple copies. Here we describe the general advantages of using RMCE to introduce genes into fly cells, the different attributes of the 2 methods, and how future work could make use of other recombinases and CRISPR/Cas9 genome editing to further enable genetic manipulation of Drosophila cells in vitro.
Asunto(s)
Drosophila/genética , Marcación de Gen/métodos , Genes de Insecto , Recombinación Genética , Transgenes , Animales , Línea Celular , Técnicas In Vitro , Recombinasas/metabolismoRESUMEN
In Drosophila, Dicer-1 binds Loquacious-PB (Loqs-PB) as its major co-factor. Previous analyses indicated that loqs mutants only partially impede miRNA processing, but the activity of minor isoforms or maternally deposited Loqs was not eliminated in these studies. We addressed this by generating a cell line from loqs-null embryos and found that only â¼40% of miRNAs showed clear Loqs dependence. Genome-wide comparison of the hairpin structure and Loqs dependence suggested that Loqs substrates are influenced by base-pairing status at the dicing site. Artificial alteration of base-pairing stability at this position in model miRNA hairpins resulted in predicted changes in Loqs dependence, providing evidence for this hypothesis. Finally, we found that evolutionarily young miRNA genes tended to be Loqs dependent. We propose that Loqs may have roles in assisting the de novo emergence of miRNA genes by facilitating dicing of suboptimal hairpin substrates.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , MicroARNs/química , MicroARNs/genética , Conformación de Ácido Nucleico , ARN Helicasas/metabolismo , Procesamiento Postranscripcional del ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleasa III/metabolismo , Animales , Emparejamiento Base/genética , Secuencia de Bases , Línea Celular , Evolución Molecular , Técnicas de Inactivación de Genes , Biblioteca de Genes , MicroARNs/metabolismo , Mutación/genética , Fenotipo , Reproducibilidad de los ResultadosRESUMEN
Vein (Vn), a ligand for the Drosophila epidermal growth factor receptor (Egfr), has a complex structure including a PEST, Ig, and EGF domain. We analyzed the structure-function relationships of Vn by assaying deletion mutants. The results show that each conserved domain influences Vn activity. A PEST deletion increases Vn potency and genetic evidence suggests that Vn is regulated by proteasomal degradation. The Ig deletion causes toxic effects not seen following expression of native Vn, but the Ig domain is not required for Vn localization or for the activation of Egfr signaling in wing vein patterning. Remarkably, when the EGF domain is deleted, Vn functions as a dominant negative ligand, implying that Vn normally physically interacts with another factor to promote its activity. We identified additional highly conserved sequences and found several regions that affect Vn potency and one that may mediate the effect of dominant negative Vn molecules. Together the results show that the activity of Vn is controlled both positively and negatively, demonstrating the existence of additional levels at which Egfr signaling can be regulated.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila/genética , Factor de Crecimiento Epidérmico/genética , Receptores ErbB/genética , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Ligandos , Datos de Secuencia Molecular , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , VenasRESUMEN
Transfection of transgenes into Drosophila cultured cells is a standard approach for studying gene function. However, the number of transgenes present in the cell following transient transfection or stable random integration varies, and the resulting differences in expression level affect interpretation. Here we developed a system for Drosophila cell lines that allows selection of cells with a single-copy transgene inserted at a specific genomic site using recombination-mediated cassette exchange (RMCE). We used the φC31 integrase and its target sites attP and attB for RMCE. Cell lines with an attP-flanked genomic cassette were transfected with donor plasmids containing a transgene of interest (UAS-x), a dihydrofolate reductase (UAS-DHFR) gene flanked by attB sequences, and a thymidine kinase (UAS-TK) gene in the plasmid backbone outside the attB sequences. In cells undergoing RMCE, UAS-x and UAS-DHFR were exchanged for the attP-flanked genomic cassette, and UAS-TK was excluded. These cells were selected using methotrexate, which requires DHFR expression, and ganciclovir, which causes death in cells expressing TK. Pure populations of cells with one copy of a stably integrated transgene were efficiently selected by cloning or mass culture in â¼6 weeks. Our results show that RMCE avoids the problems associated with current methods, where transgene number is not controlled, and facilitates the rapid generation of Drosophila cell lines in which expression from a single transgene can be studied.
Asunto(s)
Marcación de Gen/métodos , Integrasas/metabolismo , Transfección/métodos , Animales , Línea Celular , Drosophila melanogaster/genética , Femenino , Genes de Insecto , Masculino , Recombinación Genética , Técnicas de Cultivo de Tejidos , TransgenesRESUMEN
Drosophila epithelial research is at the forefront of the field; however, there are no well-characterized epithelial cell lines that could provide a complementary in vitro model for studies conducted in vivo. Here, a protocol is described that produces epithelial cell lines. The method uses genetic manipulation of oncogenes or tumor suppressors to induce embryonic primary culture cells to rapidly progress to permanent cell lines. It is, however, a general method and the type of cells that comprise a given line is not controlled experimentally. Indeed, only a small fraction of the lines produced are epithelial in character. For this reason, additional work needs to be done to develop a more robust epithelial cell-specific protocol. It is expected that Drosophila epithelial cell lines will have great utility for in vitro analysis of epithelial biology, particularly high-throughput analyses such as RNAi screens.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Drosophila melanogaster/citología , Células Epiteliales/citología , Animales , Cadherinas/metabolismo , Línea Celular , Criopreservación , Proteínas de Drosophila/genética , Células Epiteliales/metabolismo , Femenino , Genes Supresores de Tumor , Masculino , Oncogenes/genética , Óvulo/citología , Interferencia de ARN , Factores de Transcripción/genética , Transfección , Proteínas ras/genéticaRESUMEN
In vivo analysis of Drosophila melanogaster has enhanced our understanding of many biological processes, notably the mechanisms of heredity and development. While in vivo analysis of mutants has been a strength of the field, analyzing fly cells in culture is valuable for cell biological, biochemical and whole genome approaches in which large numbers of homogeneous cells are required. An efficient genetic method to derive Drosophila cell lines using expression of an oncogenic form of Ras (Ras(V12)) has been developed. Mutations in tumor suppressors, which are known to cause cell hyperproliferation in vivo, could provide another method for generating Drosophila cell lines. Here we screened Drosophila tumor suppressor mutations to test if they promoted cell proliferation in vitro. We generated primary cultures and determined when patches of proliferating cells first emerged. These cells emerged on average at 37 days in wild-type cultures. Using this assay we found that a Pten mutation had a strong effect. Patches of proliferating cells appeared on average at 11 days and the cultures became confluent in about 3 weeks, which is similar to the timeframe for cultures expressing Ras(V12). Three Pten mutant cell lines were generated and these have now been cultured for between 250 and 630 cell doublings suggesting the life of the mutant cells is likely to be indefinite. We conclude that the use of Pten mutants is a powerful means to derive new Drosophila cell lines.
Asunto(s)
Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/enzimología , Fosfohidrolasa PTEN/deficiencia , Fosfohidrolasa PTEN/metabolismo , Animales , Bioensayo , Línea Celular , Proliferación Celular , Hiperplasia , Proteínas Mutantes/metabolismo , Mutación/genética , Neoplasias/metabolismo , Neoplasias/patología , Factores de Tiempo , Proteínas Supresoras de Tumor/metabolismo , Proteínas ras/metabolismoRESUMEN
The highly conserved epidermal growth factor receptor (Egfr) pathway is required in all animals for normal development and homeostasis; consequently, aberrant Egfr signaling is implicated in a number of diseases. Genetic analysis of Drosophila melanogaster Egfr has contributed significantly to understanding this conserved pathway and led to the discovery of new components and targets. Here we used microarray analysis of third instar wing discs, in which Egfr signaling was perturbed, to identify new Egfr-responsive genes. Upregulated transcripts included five known targets, suggesting the approach was valid. We investigated the function of 29 previously uncharacterized genes, which had pronounced responses. The Egfr pathway is important for wing-vein patterning and using reverse genetic analysis we identified five genes that showed venation defects. Three of these genes are expressed in vein primordia and all showed transcriptional changes in response to altered Egfr activity consistent with being targets of the pathway. Genetic interactions with Egfr further linked two of the genes, Sulfated (Sulf1), an endosulfatase gene, and CG4096, an A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS) gene, to the pathway. Sulf1 showed a strong genetic interaction with the neuregulin-like ligand vein (vn) and may influence binding of Vn to heparan-sulfated proteoglycans (HSPGs). How Drosophila Egfr activity is modulated by CG4096 is unknown, but interestingly vertebrate EGF ligands are regulated by a related ADAMTS protein. We suggest Sulf1 and CG4096 are negative feedback regulators of Egfr signaling that function in the extracellular space to influence ligand activity.
Asunto(s)
Drosophila/metabolismo , Receptores ErbB/metabolismo , Retroalimentación Fisiológica , Transducción de Señal , Animales , Tipificación del Cuerpo/genética , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Epistasis Genética , Receptores ErbB/genética , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genotipo , Ligandos , Fenotipo , Unión Proteica , Interferencia de ARN , Sulfatasas/genética , Sulfatasas/metabolismo , Transcriptoma , Venas/metabolismo , Alas de Animales/metabolismoRESUMEN
The in vivo analysis of Drosophila using genetics, with almost a hundred year history, has produced an immense body of knowledge about biology. In vitro analysis, while arguably the poor cousin to its in vivo relative, has a utility--in biochemical analyses and in cell-based screening, for example, with RNAi. A major block to the development of in vitro analysis has been the lack of an efficient genetic method to derive cell lines from mutant Drosophila strains. We recently discovered that expression of activated Ras (Ras(V12)) provides cells in vitro with both a survival and a proliferative advantage and hence promotes the generation of cell lines. In this addendum, we provide new data describing the genesis of seven cell lines corresponding to a rumi mutant, which demonstrate that the method can be used to derive lines and study genetic mutants in vitro.