Fly Germline Research Articles

PUMILIO-mediated translational control of somatic cell cycle program promotes folliculogenesis and contributes to ovarian cancer progression.

Translational control is a fundamental mechanism regulating animal germ cell development. Gonadal somatic cells provide support and microenvironment for germ cell development to ensure fertility, yet the roles of translational control in gonadal somatic compartment remain largely undefined. We found that mouse homolog of conserved fly germline stem cell factor Pumilio, PUM1, is absent in oocytes of all growing follicles after the primordial follicle stage, instead, it is highly expressed in somatic compartments of ovaries. Global loss of Pum1, not oocyte-specific loss of Pum1, led to a significant reduction in follicular number and size as well as fertility. Whole-genome identification of PUM1 targets in ovarian somatic cells revealed an enrichment of cell proliferation pathway, including 48 key regulators of cell phase transition. Consistently granulosa cells proliferation is reduced and the protein expression of the PUM-bound Cell Cycle Regulators(PCCR) were altered accordingly in mutant ovaries, and specifically in granulosa cells. Increase in negative regulator expression and decrease in positive regulators in the mutant ovaries support a coordinated translational control of somatic cell cycle program via PUM proteins. Furthermore, postnatal knockdown, but not postnatal oocyte-specific loss, of Pum1 in Pum2 knockout mice reduced follicular growth and led to similar expression alteration of PCCR genes, supporting a critical role of PUM-mediated translational control in ovarian somatic cells for mammalian female fertility. Finally, expression of human PUM protein and its regulated cell cycle targets exhibited significant correlation with ovarian cancer and prognosis for cancer survival. Hence, PUMILIO-mediated cell cycle regulation represents an important mechanism in mammalian female reproduction and human cancer biology.

Dynamics of Asymmetric and Symmetric Divisions of Muscle Stem Cells In Vivo and on Artificial Niches

Stem cells can be maintained through symmetric cell divisions (SCDs) and asymmetric cell divisions (ACDs). How and when these divisions occur invivo in vertebrates is poorly understood. Here, we developed a clonogenic cell tracing method that demonstrates the asymmetric distribution of transcription factors along with old and new DNA in mouse muscle stem cells during skeletal muscle regeneration. Combining single-cell tracking and artificial niches exvivo, we show how cells switch from ACDs to SCDs, suggesting that they are not engaged in an obligate mode of cell division. Further, we generated SNAP-tagged histone H3-reporter mice and find that, unlike fly germline stem cells, differential fate outcomes are associated with a symmetric distribution of the H3.1 and H3.3 histone variants in mouse muscle stem cells. This versatile and efficient H3-SNAP labeling system will allow an investigation of mechanisms underlying the maintenance of epigenomic identity and plasticity in a variety of tissues.

Gene Knock-Ins in Drosophila Using Homology-Independent Insertion of Universal Donor Plasmids.

Targeted genomic knock-ins are a valuable tool to probe gene function. However, knock-in methods involving homology-directed repair (HDR) can be laborious. Here, we adapt the mammalian CRISPaint [clustered regularly interspaced short palindromic repeat (CRISPR)-assisted insertion tagging] homology-independent knock-in method for Drosophila melanogaster, which uses CRISPR/Cas9 and nonhomologous end joining to insert "universal" donor plasmids into the genome. Using this method in cultured S2R+ cells, we efficiently tagged four endogenous proteins with the bright fluorescent protein mNeonGreen, thereby demonstrating that an existing collection of CRISPaint universal donor plasmids is compatible with insect cells. In addition, we inserted the transgenesis marker 3xP3-red fluorescent protein into seven genes in the fly germ line, producing heritable loss-of-function alleles that were isolated by simple fluorescence screening. Unlike in cultured cells, insertions/deletions always occurred at the genomic insertion site, which prevents predictably matching the insert coding frame to the target gene. Despite this effect, we were able to isolate T2A-Gal4 insertions in four genes that serve as in vivo expression reporters. Therefore, homology-independent insertion in Drosophila is a fast and simple alternative to HDR that will enable researchers to dissect gene function.

The Gut Commensal Microbiome of Drosophila melanogaster Is Modified by the Endosymbiont Wolbachia.

Endosymbiotic Wolbachia bacteria and the gut microbiome have independently been shown to affect several aspects of insect biology, including reproduction, development, life span, stem cell activity, and resistance to human pathogens, in insect vectors. This work shows that Wolbachia bacteria, which reside mainly in the fly germline, affect the microbial species present in the fly gut in a lab-reared strain. Drosophila melanogaster hosts two main genera of commensal bacteria-Acetobacter and Lactobacillus. Wolbachia-infected flies have significantly reduced titers of Acetobacter. Sampling of the microbiome of axenic flies fed with equal proportions of both bacteria shows that the presence of Wolbachia bacteria is a significant determinant of the composition of the microbiome throughout fly development. However, this effect is host genotype dependent. To investigate the mechanism of microbiome modulation, the effect of Wolbachia bacteria on Imd and reactive oxygen species pathways, the main regulators of immune response in the fly gut, was measured. The presence of Wolbachia bacteria does not induce significant changes in the expression of the genes for the effector molecules in either pathway. Furthermore, microbiome modulation is not due to direct interaction between Wolbachia bacteria and gut microbes. Confocal analysis shows that Wolbachia bacteria are absent from the gut lumen. These results indicate that the mechanistic basis of the modulation of composition of the microbiome by Wolbachia bacteria is more complex than a direct bacterial interaction or the effect of Wolbachia bacteria on fly immunity. The findings reported here highlight the importance of considering the composition of the gut microbiome and host genetic background during Wolbachia-induced phenotypic studies and when formulating microbe-based disease vector control strategies. IMPORTANCEWolbachia bacteria are intracellular bacteria present in the microbiome of a large fraction of insects and parasitic nematodes. They can block mosquitos' ability to transmit several infectious disease-causing pathogens, including Zika, dengue, chikungunya, and West Nile viruses and malaria parasites. Certain extracellular bacteria present in the gut lumen of these insects can also block pathogen transmission. However, our understanding of interactions between Wolbachia and gut bacteria and how they influence each other is limited. Here we show that the presence of Wolbachia strain wMel changes the composition of gut commensal bacteria in the fruit fly. Our findings implicate interactions between bacterial species as a key factor in determining the overall composition of the microbiome and thus reveal new paradigms to consider in the development of disease control strategies.

Impact of gut microbiota on the fly's germ line.

Unlike vertically transmitted endosymbionts, which have broad effects on their host's germ line, the extracellular gut microbiota is transmitted horizontally and is not known to influence the germ line. Here we provide evidence supporting the influence of these gut bacteria on the germ line of Drosophila melanogaster. Removal of the gut bacteria represses oogenesis, expedites maternal-to-zygotic-transition in the offspring and unmasks hidden phenotypic variation in mutants. We further show that the main impact on oogenesis is linked to the lack of gut Acetobacter species, and we identify the Drosophila Aldehyde dehydrogenase (Aldh) gene as an apparent mediator of repressed oogenesis in Acetobacter-depleted flies. The finding of interactions between the gut microbiota and the germ line has implications for reproduction, developmental robustness and adaptation.

Transposition-driven genomic heterogeneity in the Drosophila brain.

Recent studies in mammals have documented the neural expression and mobility of retrotransposons and have suggested that neural genomes are diverse mosaics. We found that transposition occurs among memory-relevant neurons in the Drosophila brain. Cell type-specific gene expression profiling revealed that transposon expression is more abundant in mushroom body (MB) αβ neurons than in neighboring MB neurons. The Piwi-interacting RNA (piRNA) proteins Aubergine and Argonaute 3, known to suppress transposons in the fly germline, are expressed in the brain and appear less abundant in αβ MB neurons. Loss of piRNA proteins correlates with elevated transposon expression in the brain. Paired-end deep sequencing identified more than 200 de novo transposon insertions in αβ neurons, including insertions into memory-relevant loci. Our observations indicate that genomic heterogeneity is a conserved feature of the brain.

The enigmatic cytoophidium: Compartmentation of CTP synthase via filament formation

The enigmatic cytoophidium: Compartmentation of CTP synthase via filament formation

Drosophila Translational Elongation Factor-1γ Is Modified in Response to DOA Kinase Activity and Is Essential for Cellular Viability

Drosophila translational elongation factor-1gamma (EF1gamma) interacts in the yeast two-hybrid system with DOA, the LAMMER protein kinase of Drosophila. Analysis of mutant EF1gamma alleles reveals that the locus encodes a structurally conserved protein essential for both organismal and cellular survival. Although no genetic interactions were detected in combinations with mutations in EF1alpha, an EF1gamma allele enhanced mutant phenotypes of Doa alleles. A predicted LAMMER kinase phosphorylation site conserved near the C terminus of all EF1gamma orthologs is a phosphorylation site in vitro for both Drosophila DOA and tobacco PK12 LAMMER kinases. EF1gamma protein derived from Doa mutant flies migrates with altered mobility on SDS gels, consistent with it being an in vivo substrate of DOA kinase. However, the aberrant mobility appears to be due to a secondary protein modification, since the mobility of EF1gamma protein obtained from wild-type Drosophila is unaltered following treatment with several nonspecific phosphatases. Expression of a construct expressing a serine-to-alanine substitution in the LAMMER kinase phosphorylation site into the fly germline rescued null EF1gamma alleles but at reduced efficiency compared to a wild-type construct. Our data suggest that EF1gamma functions in vital cellular processes in addition to translational elongation and is a LAMMER kinase substrate in vivo.

Translational Repression of Cyclin E Prevents Precocious Mitosis and Embryonic Gene Activation during C. elegans Meiosis

Germ cells, the cells that give rise to sperm and egg, maintain the potential to recreate all cell types in a new individual. This wide developmental potential, or totipotency, is manifested in unusual tumors called teratomas, in which germ cells undergo somatic differentiation. Although recent studies have implicated RNA regulation, the mechanism that normally prevents the loss of germ cell identity remains unexplained. In C. elegans, a teratoma is induced in the absence of the conserved RNA-binding protein GLD-1. Here, we demonstrate that GLD-1 represses translation of CYE-1/cyclin E during meiotic prophase, which prevents germ cells from re-entering mitosis and inducing embryonic-like transcription. We describe a mechanism that prevents precocious mitosis in germ cells undergoing meiosis, propose that this mechanism maintains germ cell identity by delaying the onset of embryonic gene activation until after fertilization, and provide a paradigm for the possible origin of human teratomas.

A Distinct Small RNA Pathway Silences Selfish Genetic Elements in the Germline

In the Drosophila germline, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous selfish genetic elements such as retrotransposons and repetitive sequences. Whereas small interfering RNAs (siRNAs) derive from both the sense and antisense strands of their double-stranded RNA precursors, rasiRNAs arise mainly from the antisense strand. rasiRNA production appears not to require Dicer-1, which makes microRNAs (miRNAs), or Dicer-2, which makes siRNAs, and rasiRNAs lack the 2',3' hydroxy termini characteristic of animal siRNA and miRNA. Unlike siRNAs and miRNAs, rasiRNAs function through the Piwi, rather than the Ago, Argonaute protein subfamily. Our data suggest that rasiRNAs protect the fly germline through a silencing mechanism distinct from both the miRNA and RNA interference pathways.

Repression of hybrid dysgenesis in Drosophila melanogaster by combinations of telomeric P-element reporters and naturally occurring P elements.

In Drosophila melanogaster, hybrid dysgenesis occurs in the germline of flies produced by crosses between females lacking P elements and males carrying 25-55 P elements. We have previously shown that a complete maternally inherited repression of P transposition in the germline (P cytotype) can be elicited by only two autonomous P elements located at the X chromosome telomere (cytological site 1A). We have tested whether P transgenes at 1A, unable to code for a P-repressor, may contribute to the repression of P elements. Females carrying a P-lacZ transgene at 1A ["P-lacZ(1A)"], crossed with P males, do not repress dysgenic sterility in their progeny. However, these P-lacZ(1A) insertions, maternally or paternally inherited, contribute to P-element repression when they are combined with other regulatory P elements. This combination effect is not seen when the P-lacZ transgene is located in pericentromeric heterochromatin or in euchromatin; however a P-w,ry transgene located at the 3R chromosome telomere exhibits the combination effect. The combination effect with the P-lacZ(1A) transgene is impaired by a mutant Su(var)205 allele known to impair the repression ability of the autonomous P elements at 1A. We hypothesized that the combination effect is due to modification of the chromatin structure or nuclear location of genomic P elements.

Invertebrate retroviruses: ZAM a new candidate in D.melanogaster

ZAM, a new retroelement of Drosophila melanogaster, was identified as a mutational insertion at the white locus. It displays all the structural features of a vertebrate retrovirus. Its three open reading frames encode predicted products resembling the products of the gag, pol and env genes of retroviruses. Its transcription gives rise to an 8.6 kb full-length RNA and a 1.7 kb spliced message for the env gene. The latter encodes an envelope protein that is typical of elements having an extracellular phase of the life cycle. The identification of a ZAM envelope retrogene provides evidence that ZAM is mobilized through a reverse trancriptional process in the germ line of flies. We report that ZAM is distributed differently among D.melanogaster strains. Two stocks out of >15 tested display a ZAM high copy number, with numerous copies distributed on chromosomal arms. This high copy number is associated with a high transcriptional rate of ZAM. The existence of these two categories of strains offers a new genetic system in which the properties of a potential invertebrate retrovirus can be tested.

Homeo Boxes in the Study of Development

The body plan of Drosophila is determined to a large extent by homeotic genes, which specify the identity and spatial arrangement of the body segments. Homeotic genes share a characteristic DNA segment, the homeo box, which encodes a defined domain of the homeotic proteins. The homeo domain seems to mediate the binding to specific DNA sequences, whereby the homeotic proteins exert a gene regulatory function. By isolating the normal Antennapedia gene, fusing its protein-coding sequences to an inducible promoter, and reintroducing this fusion gene into the germline of flies, it has been possible to transform head structures into thoracic structures and to alter the body plan in a predicted way. Sequence homologies suggest that similar genetic mechanisms may control development in higher organisms.