RESUMO
Notch signaling and epigenetic factors are known to play critical roles in regulating tissue homeostasis in most multicellular organisms, but how Notch signaling coordinates with epigenetic modulators to control differentiation remains poorly understood. Here, we identify heterochromatin protein 1c (HP1c) as an essential epigenetic regulator of gut homeostasis in Drosophila. Specifically, we observe that HP1c loss-of-function phenotypes resemble those observed after Notch signaling perturbation and that HP1c interacts genetically with components of the Notch pathway. HP1c represses the transcription of Notch target genes by directly interacting with Suppressor of Hairless (Su(H)), the key transcription factor of Notch signaling. Moreover, phenotypes caused by depletion of HP1c in Drosophila can be rescued by expressing human HP1γ, suggesting that HP1γ functions similar to HP1c in Drosophila. Taken together, our findings reveal an essential role of HP1c in normal development and gut homeostasis by suppressing Notch signaling.
Assuntos
Proteínas de Drosophila , Animais , Proteínas Cromossômicas não Histona/genética , Drosophila/genética , Proteínas de Drosophila/genética , Heterocromatina , Homeostase , Humanos , Receptores Notch/genéticaRESUMO
The niche controls stem cell self-renewal and differentiation in animal tissues. Although the exocyst is known to be important for protein membrane trafficking and secretion, its role in stem cells and niches has never been reported. Here, this study shows that the exocyst functions in the niche to promote germline stem cell (GSC) progeny differentiation in the Drosophila ovary by directly regulating EGFR membrane trafficking and signaling. Inactivation of exocyst components in inner germarial sheath cells, which form the differentiation niche, causes a severe GSC differentiation defect. The exocyst is required for maintaining niche cells and preventing BMP signaling in GSC progeny by promoting EGFR membrane targeting and signaling through direct association with EGFR. Finally, it is also required for EGFR membrane targeting, recycling and signaling in human cells. Therefore, this study reveals a novel function of the exocyst in niche cells to promote stem cell progeny differentiation by directly controlling EGFR membrane trafficking and signaling in vivo, and also provides important insight into how the niche controls stem cell progeny differentiation at the molecular level.
Assuntos
Diferenciação Celular , Proteínas de Drosophila/metabolismo , Receptores ErbB/metabolismo , Células Germinativas/citologia , Receptores de Peptídeos de Invertebrados/metabolismo , Nicho de Células-Tronco , Células-Tronco/fisiologia , Proteínas de Transporte Vesicular/fisiologia , Animais , Animais Geneticamente Modificados , Diferenciação Celular/genética , Membrana Celular/metabolismo , Autorrenovação Celular/genética , Células Cultivadas , Drosophila , Proteínas de Drosophila/fisiologia , Receptores ErbB/fisiologia , Feminino , Proteínas de Ligação ao GTP/fisiologia , Células Germinativas/metabolismo , Células HEK293 , Células HeLa , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/fisiologia , Ovário/citologia , Ovário/metabolismo , Transporte Proteico/genética , Receptores de Peptídeos de Invertebrados/fisiologia , Nicho de Células-Tronco/genética , Células-Tronco/citologia , Proteínas de Transporte Vesicular/genéticaRESUMO
CRISPR/Cas9-based transcriptional activation (CRISPRa) has recently emerged as a powerful and scalable technique for systematic overexpression genetic analysis in Drosophila melanogaster We present flySAM, a potent tool for in vivo CRISPRa, which offers major improvements over existing strategies in terms of effectiveness, scalability, and ease of use. flySAM outperforms existing in vivo CRISPRa strategies and approximates phenotypes obtained using traditional Gal4-UAS overexpression. Moreover, because flySAM typically requires only a single sgRNA, it dramatically improves scalability. We use flySAM to demonstrate multiplexed CRISPRa, which has not been previously shown in vivo. In addition, we have simplified the experimental use of flySAM by creating a single vector encoding both the UAS:Cas9-activator and the sgRNA, allowing for inducible CRISPRa in a single genetic cross. flySAM will replace previous CRISPRa strategies as the basis of our growing genome-wide transgenic overexpression resource, TRiP-OE.
Assuntos
Animais Geneticamente Modificados , Sistemas CRISPR-Cas , Proteínas de Drosophila , Regulação da Expressão Gênica/genética , Fatores de Transcrição , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Drosophila melanogaster , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
Drosophila ovary is recognized as one of the best model systems to study stem cell biology in vivo. We had previously identified an autonomous role of the histone H1 in germline stem cell (GSC) maintenance. Here, we found that histone H1 depletion in escort cells (ECs) resulted in an increase of spectrosome-containing cells (SCCs), an ovary tumor-like phenotype. Further analysis showed that the Dpp pathway is excessively activated in these SCC cells, while the expression of bam is attenuated. In the H1-depleted ECs, both transposon activity and DNA damage had increased dramatically, followed by EC apoptosis, which is consistent with the role of H1 in other somatic cells. Surprisingly, H1-depleted ECs acquired cap cell characteristics including dpp expression, and the resulting abnormal Dpp level inhibits SCC further differentiation. Most interestingly, double knockdown of H1 and dpp in ECs can reduce the number of SCCs to the normal level, indicating that the additional Dpp secreted by ECs contributes to the germline tumor. Taken together, our findings indicate that histone H1 is an important epigenetic factor in controlling EC characteristics and a key suppressor of germline tumor.
Assuntos
Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Células Germinativas/metabolismo , Células Germinativas/patologia , Histonas/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Animais , Apoptose , Contagem de Células , Dano ao DNA , Elementos de DNA Transponíveis/genética , Feminino , Técnicas de Silenciamento de Genes , Modelos Biológicos , Fenótipo , Transdução de Sinais , Transcrição Gênica , Regulação para CimaRESUMO
The ability to engineer genomes in a specific, systematic, and cost-effective way is critical for functional genomic studies. Recent advances using the CRISPR-associated single-guide RNA system (Cas9/sgRNA) illustrate the potential of this simple system for genome engineering in a number of organisms. Here we report an effective and inexpensive method for genome DNA editing in Drosophila melanogaster whereby plasmid DNAs encoding short sgRNAs under the control of the U6b promoter are injected into transgenic flies in which Cas9 is specifically expressed in the germ line via the nanos promoter. We evaluate the off-targets associated with the method and establish a Web-based resource, along with a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Finally, we discuss the advantages of our method in comparison with other recently published approaches.
Assuntos
Sistemas CRISPR-Cas/genética , Drosophila melanogaster/genética , Engenharia Genética/métodos , Genômica/métodos , Células Germinativas , Animais , Animais Geneticamente Modificados , Bases de Dados Genéticas , Proteínas de Drosophila/genética , Mutagênese/genética , Regiões Promotoras Genéticas/genética , Proteínas de Ligação a RNA/genéticaRESUMO
N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic mRNA, is installed by a multi-component writer complex; however, the exact roles of each component remain poorly understood. Here we show that a potential E3 ubiquitin ligase Hakai colocalizes and interacts with other m6A writer components, and Hakai mutants exhibit typical m6A pathway defects in Drosophila, such as lowered m6A levels in mRNA, aberrant Sxl alternative splicing, wing and behavior defects. Hakai, Vir, Fl(2)d and Flacc form a stable complex, and disruption of either Hakai, Vir or Fl(2)d led to the degradation of the other three components. Furthermore, MeRIP-seq indicates that the effective m6A modification is mostly distributed in 5' UTRs in Drosophila, in contrast to the mammalian system. Interestingly, we demonstrate that m6A modification is deposited onto the Sxl mRNA in a sex-specific fashion, which depends on the m6A writer. Together, our work not only advances the understanding of mechanism and regulation of the m6A writer complex, but also provides insights into how Sxl cooperate with the m6A pathway to control its own splicing.
Assuntos
Adenosina/análogos & derivados , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Regiões 5' não Traduzidas/genética , Adenosina/metabolismo , Processamento Alternativo/genética , Animais , Sequência de Bases , Comportamento Animal , Códon de Iniciação/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Proteínas de Fluorescência Verde/metabolismo , Masculino , Metilação , Mutação/genética , Ligação Proteica , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Caracteres Sexuais , Ubiquitina-Proteína Ligases/genética , Asas de Animais/metabolismoRESUMO
Mechanoreceptor cells develop a specialized cytoskeleton that plays structural and sensory roles at the site of mechanotransduction. However, little is known about how the cytoskeleton is organized and formed. Using electron tomography and live-cell imaging, we resolve the 3D structure and dynamics of the microtubule-based cytoskeleton in fly campaniform mechanosensory cilia. Investigating the formation of the cytoskeleton, we find that katanin p60-like 1 (kat-60L1), a neuronal type of microtubule-severing enzyme, serves two functions. First, it amplifies the mass of microtubules to form the dense microtubule arrays inside the sensory cilia. Second, it generates short microtubules that are required to build the nanoscopic cytoskeleton at the mechanotransduction site. Additional analyses further reveal the functional roles of Patronin and other potential factors in the local regulatory network. In all, our results characterize the specialized cytoskeleton in fly external mechanosensory cilia at near-molecular resolution and provide mechanistic insights into how it is formed.
Assuntos
Proteínas de Drosophila/metabolismo , Katanina/metabolismo , Mecanotransdução Celular , Animais , Polaridade Celular , Drosophila melanogaster/metabolismo , Drosophila melanogaster/ultraestrutura , Extremidades/fisiologia , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Modelos Biológicos , Organelas/metabolismo , Organelas/ultraestrutura , Receptores de Superfície Celular/metabolismoRESUMO
The flySAM/CRISPRa system has recently emerged as a powerful tool for gain-of-function studies in Drosophila melanogaster This system includes Gal4/UAS-driven dCas9 activators and U6 promoter-controlled sgRNA. Having established dCas9 activators superior to other combinations, to further enhance the efficiency of the targeting activators we systematically optimized the parameters of the sgRNA. Interestingly, the most efficient sgRNAs were found to accumulate in the region from -150bp to -450bp upstream of the transcription start site (TSS), and the activation efficiency showed a strong positive correlation with the GC content of the sgRNA targeting sequence. In addition, the target region is dominant to the GC content, as sgRNAs targeting areas beyond -600bp from the TSS lose efficiency even when containing 75% GC. Surprisingly, when comparing the activities of sgRNAs targeting to either DNA strand, sgRNAs targeting to the non-template strand outperform those complementary to the template strand, both in cells and in vivo In summary, we define criteria for sgRNA design which will greatly facilitate the application of CRISPRa in gain-of-function studies.
Assuntos
Drosophila melanogaster , Drosophila , Animais , Composição de Bases , Sistemas CRISPR-Cas , Drosophila/genética , Drosophila melanogaster/genética , Regiões Promotoras Genéticas , RNA Guia de Cinetoplastídeos/genética , Sítio de Iniciação de TranscriçãoRESUMO
Polyploid cells endoreplicate their DNA through a modified cell cycle that skips mitosis as part of their differentiation programs. Upon cell-cycle exit and differentiation, non-centrosomal sites govern microtubule distribution in most cells. Little is known on how polyploid cells, differentiated but cycling, organize their microtubules. We show that microtubules in Drosophila adipocytes and other polyploid tissues form a dense perinuclear cortex responsible for nuclear size and position. Confirming a relation between this perinuclear cortex and the polyploid endocycle, polyploidization of normally diploid cells was sufficient for cortex formation. A critical component of the perinuclear microtubule organizer (pnMTOC) is Shot, absence of which caused collapse of the perinuclear network into a condensed organizer through kinesin-dependent microtubule sliding. Furthermore, this ectopic organizer was capable of directing partial assembly of a deeply disruptive cytokinesis furrow. In all, our study revealed the importance of perinuclear microtubule organization for stability of endocycling Drosophila cells.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Katanina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/fisiologia , Poliploidia , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citocinese , Proteínas de Drosophila/genética , Feminino , Katanina/genética , Masculino , Proteínas dos Microfilamentos/genética , Proteínas Associadas aos Microtúbulos/genética , Fuso AcromáticoRESUMO
Much of our knowledge about the mechanisms underlying biological processes relies on genetic approaches, whereby gene activity is reduced and the phenotypic consequences of perturbation are analyzed in detail. For functional genomic studies, a specific, systematic, and cost-effective manner is critical. Transgenic RNAi system is the top priority choice to study gene functions due to its simple and practical characteristics in Drosophila. We established a novel system that works well in both soma and germ cells which is efficient and specific. With this system, we can precisely and efficiently modulate highly expressed genes, and simultaneously knock down multiple genes in one step. In this study, we provide a detailed protocol of the pNP system, which replaces other transgenic systems, and expect it can provide some help to researchers who are using this system.
RESUMO
Adequate protein intake is crucial for the survival and well-being of animals. How animals assess prospective protein sources and ensure dietary amino acid intake plays a critical role in protein homeostasis. By using a quantitative feeding assay, we show that three amino acids, L-glutamate (L-Glu), L-alanine (L-Ala) and L-aspartate (L-Asp), but not their D-enantiomers or the other 17 natural L-amino acids combined, rapidly promote food consumption in the fruit fly Drosophila melanogaster. This feeding-promoting effect of dietary amino acids is independent of mating experience and internal nutritional status. In vivo and ex vivo calcium imagings show that six brain neurons expressing diuretic hormone 44 (DH44) can be rapidly and directly activated by these amino acids, suggesting that these neurons are an amino acid sensor. Genetic inactivation of DH44+ neurons abolishes the increase in food consumption induced by dietary amino acids, whereas genetic activation of these neurons is sufficient to promote feeding, suggesting that DH44+ neurons mediate the effect of dietary amino acids to promote food consumption. Single-cell transcriptome analysis and immunostaining reveal that a putative amino acid transporter, CG13248, is enriched in DH44+ neurons. Knocking down CG13248 expression in DH44+ neurons blocks the increase in food consumption and eliminates calcium responses induced by dietary amino acids. Therefore, these data identify DH44+ neuron as a key sensor to detect amino acids and to enhance food intake via a putative transporter CG13248. These results shed critical light on the regulation of protein homeostasis at organismal levels by the nervous system.
Assuntos
Aminoácidos/metabolismo , Drosophila/fisiologia , Ingestão de Alimentos , Alanina/metabolismo , Alanina/farmacologia , Aminoácidos/farmacologia , Animais , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacologia , Cálcio/metabolismo , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Comportamento Alimentar/efeitos dos fármacos , Feminino , Ácido Glutâmico/metabolismo , Ácido Glutâmico/farmacologia , Hormônios de Inseto/genética , Hormônios de Inseto/metabolismo , Masculino , Neurônios/metabolismo , Estado Nutricional , Interferência de RNA , RNA Interferente Pequeno/metabolismo , TranscriptomaRESUMO
Being relatively simple and practical, Drosophila transgenic RNAi is the technique of top priority choice to quickly study genes with pleiotropic functions. However, drawbacks have emerged over time, such as high level of false positive and negative results. To overcome these shortcomings and increase efficiency, specificity and versatility, we develop a next generation transgenic RNAi system. With this system, the leaky expression of the basal promoter is significantly reduced, as well as the heterozygous ratio of transgenic RNAi flies. In addition, it has been first achieved to precisely and efficiently modulate highly expressed genes. Furthermore, we increase versatility which can simultaneously knock down multiple genes in one step. A case illustration is provided of how this system can be used to study the synthetic developmental effect of histone acetyltransferases. Finally, we have generated a collection of transgenic RNAi lines for those genes that are highly homologous to human disease genes.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regiões Promotoras Genéticas/genética , Interferência de RNA , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imuno-Histoquímica , Masculino , Microscopia Confocal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismoRESUMO
Recent studies of the Cas9/sgRNA system in Drosophila melanogaster genome editing have opened new opportunities to generate site-specific mutant collections in a high-throughput manner. However, off-target effects of the system are still a major concern when analyzing mutant phenotypes. Mutations converting Cas9 to a DNA nickase have great potential for reducing off-target effects in vitro. Here, we demonstrated that injection of two plasmids encoding neighboring offset sgRNAs into transgenic Cas9(D10A) nickase flies efficiently produces heritable indel mutants. We then determined the effective distance between the two sgRNA targets and their orientations that affected the ability of the sgRNA pairs to generate mutations when expressed in the transgenic nickase flies. Interestingly, Cas9 nickase greatly reduces the ability to generate mutants with one sgRNA, suggesting that the application of Cas9 nickase and sgRNA pairs can almost avoid off-target effects when generating indel mutants. Finally, a defined piwi mutant allele is generated with this system through homology-directed repair. However, Cas9(D10A) is not as effective as Cas9 in replacing the entire coding sequence of piwi with two sgRNAs.
Assuntos
Desoxirribonuclease I/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Animais , Animais Geneticamente Modificados/metabolismo , Proteínas Argonautas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Reparo do DNA , Desoxirribonuclease I/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Embrião não Mamífero/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Mutagênese , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in Drosophila melanogaster. However, single-guide RNA (sgRNA) parameters affecting the specificity and efficiency of the system in flies are still not clear. Here, we found that off-target effects did not occur in regions of genomic DNA with three or more nucleotide mismatches to sgRNAs. Importantly, we document for a strong positive correlation between mutagenesis efficiency and sgRNA GC content of the six protospacer-adjacent motif-proximal nucleotides (PAMPNs). Furthermore, by injecting well-designed sgRNA plasmids at the optimal concentration we determined, we could efficiently generate mutations in four genes in one step. Finally, we generated null alleles of HP1a using optimized parameters through homology-directed repair and achieved an overall mutagenesis rate significantly higher than previously reported. Our work demonstrates a comprehensive optimization of sgRNA and promises to vastly simplify CRISPR/Cas9 experiments in Drosophila.