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Proper formation and specification of Primordial Germ Cells (PGCs) is of special significance as they gradually transform into Germline Stem Cells (GSCs) that are ultimately responsible for generating the gametes. Intriguingly, not only the PGCs constitute the only immortal cell type but several specific determinants also underlying PGC specification such as Vasa, Nanos and Germ-cell-less are conserved through evolution. In Drosophila melanogaster, PGC formation and specification depends on two independent factors, the maternally deposited specialized cytoplasm (or germ plasm) enriched in germline determinants, and the mechanisms that execute the even partitioning of these determinants between the daughter cells. Prior work has shown that Oskar protein is necessary and sufficient to assemble the functional germ plasm, whereas centrosomes associated with the nuclei that invade the germ plasm are responsible for its equitable distribution. Our recent data suggests that Caspar, the Drosophila orthologue of human Fas-associated factor-1 (FAF1) is a novel regulator that modulates both mechanisms that underlie the determination of PGC fate. Consistently, early blastoderm embryos derived from females compromised for caspar display reduced levels of Oskar and defective centrosomes.

Drosophila pseudoobscura and D. persimilis are a sister species pair that have been used as a model for studies of reproductive isolation and speciation for almost 100 years owing to their close evolutionary history, well characterized genetic differences, and overlapping geographic distribution. There are extensive analyses of both pre- and post-zygotic isolation, including studies of courtship divergence, conspecific sperm precedence (CSP) and how reinforcement by natural selection may or may not act to strengthen isolation in sympatry. Post-zygotic analyses explore the underlying mechanics of reproductive isolation; how inversions may give rise to initial speciation events and misexpression of key genes typically found within inversion regions render hybrid offspring unfit or inviable. We aim here to present a history of studies of reproductive isolation between this species pair, looking at how the field has developed over the last century and identifying the open questions and gaps within the literature.

The in situ hybridization chain reaction (isHCR) is a powerful method for visualizing mRNA in many species. We present a rapid isHCR method for Drosophila embryos and ovaries. Ethylene carbonate was added to the hybridization buffer to facilitate the hybridization reaction, and a modified short hairpin DNA was used in the amplification reaction; these modifications decreased the RNA staining time from 3 days to 1 day. This method is compatible with immunohistochemistry and can detect multiple mRNAs. The proposed method could significantly reduce staining time for Drosophila researchers using isHCR.

One hundred years ago, two reports appeared of tetraploid D. melanogaster females - curiosities that had never been seen before. The authors, Calvin Bridges and Lilian Morgan, were among the famed founders of fly genetics in T.H. Morgan's lab at Columbia University. Sadly, their findings have faded into the fog of ancient fly lore. This review exhumes those relics in order to offer modern fly-pushers some possible avenues for polyploid research. That subfield is undergoing a revival that may interest them.

The drugs mifepristone and rapamycin were compared for their relative ability to increase the life span of mated female Drosophila melanogaster. Titration of rapamycin indicated an optimal concentration of approximately 50 μM, which increased median life span here by average +81%. Meta-analysis of previous mifepristone titrations indicated an optimal concentration of approximately 466 μM, which increased median life span here by average +114%. Combining mifepristone with various concentrations of rapamycin did not produce further increases in life span, and instead reduced life span relative to either drug alone. Assay of maximum midgut diameter indicated that rapamycin was equally efficacious as mifepristone in reducing mating-induced midgut hypertrophy. The mito-QC mitophagy reporter is a previously described green fluorescent protein (GFP)-mCherry fusion protein targeted to the outer mitochondrial membrane. Inhibition of GFP fluorescence by the acidic environment of the autophagolysosome yields an increased red/green fluorescence ratio indicative of increased mitophagy. Creation of a multi-copy mito-QC reporter strain facilitated assay in live adult flies, as well as in dissected midgut tissue. Mifepristone was equally efficacious as rapamycin in activating the mito-QC mitophagy reporter in the adult female fat-body and midgut. The data suggest that mifepristone and rapamycin act through a common pathway to increase mated female Drosophila life span, and implicate increased mitophagy and decreased midgut hypertrophy in that pathway.

Talaromycosis, caused by Talaromyces marneffei (T. marneffei, formerly known as Penicillium marneffei), is an opportunistic invasive mycosis endemic in tropical and subtropical areas of Asia with high mortality rate. Despite various infection models established to study the immunological interaction between T. marneffei and the host, the pathogenicity of this fungus is not yet fully understood. So far, Drosophila melanogaster, a well-established genetic model organism to study innate immunity, has not been used in related research on T. marneffei. In this study, we provide the initial characterization of a systemic infection model of T. marneffei in the D. melanogaster host. Survival curves and fungal loads were tested as well as Toll pathway activation was quantified by RT-qPCR of several antimicrobial peptide (AMP) genes including Drosomycin, Metchnikowin, and Bomanin Short 1. We discovered that whereas most wild-type flies were able to overcome the infection, MyD88 or Toll mutant flies failed to prevent fungal dissemination and proliferation and ultimately succumbed to this challenge. Unexpectedly, the induction of classical Toll pathway activation readouts, Drosomycin and Bomanin Short 1, by live or killed T. marneffei was quite limited in wild-type flies, suggesting that the fungus largely escapes detection by the systemic immune system. This unusual situation of a poor systemic activation of the Toll pathway and a strong susceptibility phenotype of MyD88/Toll might be accounted for by a requirement for this host defence in only specific tissues, a hypothesis that remains to be rigorously tested.

In situ hybridization techniques are powerful methods for exploring gene expression in a wide range of biological contexts, providing spatial information that is most often lost in traditional biochemical techniques. However, many in situ hybridization methods are costly and time-inefficient, particularly for screening-based projects that follow on from single-cell RNA sequencing data, which rely on of tens of custom-synthetized probes against each specific RNA of interest. Here we provide an optimized pipeline for Hybridization Chain Reaction (HCR)-based RNA visualization, including an open-source code for optimized probe design. Our method achieves high specificity and sensitivity with the option of multiplexing using only five pairs of probes, which greatly lowers the cost and time of the experiment. These features of our HCR protocol are particularly useful and convenient for projects involving screening several genes at medium throughput, especially as the method include an amplification step, which makes the signal readily visible at low magnification imaging.

To identify genes required for brain growth, we took an RNAi knockdown reverse genetic approach in Drosophila. One potential candidate isolated from this effort is the anti-lipogenic gene adipose (adp). Adp has an established role in the negative regulation of lipogenesis in the fat body of the fly and adipose tissue in mammals. While fat is key to proper development in general, adp has not been investigated during brain development. Here, we found that RNAi knockdown of adp in neuronal stem cells and neurons results in reduced brain lobe volume and sought to replicate this with a mutant fly. We generated a novel adp mutant that acts as a loss-of-function mutant based on buoyancy assay results. We found that despite a change in fat content in the body overall and a decrease in the number of larger (>5 µm) brain lipid droplets, there was no change in the brain lobe volume of mutant larvae. Overall, our work describes a novel adp mutant that can functionally replace the long-standing adp⁶⁰ mutant and shows that the adp gene has no obvious involvement in brain growth.

Adenosine-to-inosine (A-to-I) RNA editing recodes the genome and confers flexibility for the organisms to adapt to the environment. It is believed that RNA recoding sites are well suited for facilitating adaptive evolution by increasing the proteomic diversity in a temporal-spatial manner. The function and essentiality of a few conserved recoding sites are recognized. However, the experimentally discovered functional sites only make up a small corner of the total sites, and there is still the need to expand the repertoire of such functional sites with bioinformatic approaches. In this study, we define a new category of RNA editing sites termed 'conserved editing with non-conserved recoding' and systematically identify such sites in Drosophila editomes, figuring out their selection pressure and signals of adaptation at inter-species and intra-species levels. Surprisingly, conserved editing sites with non-conserved recoding are not suppressed and are even slightly overrepresented in Drosophila. DNA mutations leading to such cases are also favoured during evolution, suggesting that the function of those recoding events in different species might be diverged, specialized, and maintained. Finally, structural prediction suggests that such recoding in potassium channel Shab might increase ion permeability and compensate the effect of low temperature. In conclusion, conserved editing with non-conserved recoding might be functional as well. Our study provides novel aspects in considering the adaptive evolution of RNA editing sites and meanwhile expands the candidates of functional recoding sites for future validation.

The Drosophila melanogaster brain is a complex organ with various cell types, orchestrating the development, physiology, and behaviors of the fly. While each cell type in Drosophila brain is known to express a unique gene set, their complete genetic profile is still unknown. Advances in the RNA sequencing techniques at single-cell resolution facilitate identifying novel cell type markers and/or re-examining the specificity of the available ones. In this study, exploiting a single-cell RNA sequencing data of Drosophila optic lobe, we categorized the cells based on their expression pattern for known markers, then the genes with enriched expression in astrocytes were identified. CG11000 was identified as a gene with a comparable expression profile to the Eaat1 gene, an astrocyte marker, in every individual cell inside the Drosophila optic lobe and midbrain, as well as in the entire Drosophila brain throughout its development. Consistent with our bioinformatics data, immunostaining of the brains dissected from transgenic adult flies showed co-expression of CG11000 with Eaat1 in a set of single cells corresponding to the astrocytes in the Drosophila brain. Physiologically, inhibiting CG11000 through RNA interference disrupted the normal development of male D. melanogaster, while having no impact on females. Expression suppression of CG11000 in adult flies led to decreased locomotion activity and also shortened lifespan specifically in astrocytes, indicating the gene's significance in astrocytes. We designated this gene as 'deathstar' due to its crucial role in maintaining the star-like shape of glial cells, astrocytes, throughout their development into adult stage.