In Drosophila, the piRNA cluster, flamenco, produces most of the piRNAs (PIWI-interacting RNAs) that silence transposable elements in the somatic follicle cells during oogenesis. These piRNAs are thought to be processed from a long single-stranded precursor transcript. Here, we demonstrate that flamenco transcription is initiated from an RNA polymerase II promoter containing an initiator motif (Inr) and downstream promoter element (DPE) and requires the transcription factor, Cubitus interruptus. We show that the flamenco precursor transcript undergoes differential alternative splicing to generate diverse RNA precursors that are processed to piRNAs. Our data reveal dynamic processing steps giving rise to piRNA cluster precursors.

The linker of nucleoskeleton and cytoskeleton (LINC) complex is an evolutionarily well-conserved protein bridge connecting the cytoplasmic and nuclear compartments across the nuclear membrane. While recent data support its function in nuclear morphology and meiosis, its involvement in chromatin organisation has not been studied in plants. Here, 3D imaging methods have been used to investigate nuclear morphology and chromatin organisation in interphase nuclei of the model plant Arabidopsis thaliana in which heterochromatin clusters in conspicuous chromatin domains called chromocentres. Chromocentres form a repressive chromatin environment contributing to transcriptional silencing of repeated sequences, a general mechanism needed for genome stability. Quantitative measurements of the 3D position of chromocentres indicate their close proximity to the nuclear periphery but that their position varies with nuclear volume and can be altered in specific mutants affecting the LINC complex. Finally, we propose that the plant LINC complex contributes to proper heterochromatin organisation and positioning at the nuclear periphery, since its alteration is associated with the release of transcriptional silencing as well as decompaction of heterochromatic sequences.

The expression of fibroblast growth factor (FGF) 1, a potent neurotrophic factor, increases during differentiation and remains high in adult neuronal tissues. To examine the importance of this expression on the neuronal phenotype, we have used PC12 cells, a model to study FGF-induced neuronal differentiation. After demonstrating that FGF1 and FGF2 are synthesized by PC12 cells, we investigated if FGF1 expression could be a key element in differentiation. Using the cell signaling pathway to determine the effects of FGF1 alone, FGF1 plus heparin, or a mutated FGF1, we showed an activation to the same extent of mitogen-activated protein (MAP) kinase kinase and MAP kinase (extracellular regulated kinase 1). However, only FGF1 plus heparin could promote PC12 cell differentiation. Thus, the MAP kinase pathway is insufficient to promote differentiation. Analysis of the PC12 cells after the addition of FGF1 plus heparin or FGF2 demonstrated a significant increase in the level of FGF1 expression with the same time course as the appearance of the neuritic extensions. Transfection experiments were performed to enhance constitutivly or after dexamethasone induction the level of FGF1 expression. The degree of differentiation achieved by the cells correlated directly with the amount of FGF1 expressed. The MAP kinase pathway did not appear to be involved. Interestingly, a 5-fold increase in FGF1 in constitutive transfected cells extended dramatically their survival in serum-free medium, suggesting that the rise of FGF1 synthesis during neuronal differentiation is probably linked to their ability to survive in the adult. All of these data demonstrate that, in contrast to the MAP kinase cascade, FGF1 expression is sufficient to induce in PC12 cells both differentiation and survival. It also shows that auto-and trans-activation of FGF1 expression is involved in the differentiation process stimulated by exogenous FGFs through a new pathway which remains to be characterized. FGF11 and 2 are widely distributed in the peripheral and central nervous systems in the adult. In rat brain, FGF2 is present in most neurons within the cerebral cortex (1), hippocampus (2), and cerebellum (3). High levels of FGF1 expression have been observed in motor neurons, primary sensory neurons, and retinal ganglion neurons (4, 5). In chick brain, the expression of FGF1 is developmentally regulated (6). In bovine and rat embryonic retina, all neuronal layers express FGF1 with an appearance corresponding to their sequential differentiation (7, 8). In rat, the level of FGF1 expression remains uniformly low throughout the embryonic period until postnatal day 7. Thereafter, it increases rapidly, reaching a maximum in the adult retina. In the intermediate central nervous system, subclasses of FGF receptors appear to be down-regulated during development (9), and during retinal embryonic development, the expression of FGFR1 and FGFR2 follows the retinal layering (10). These patterns of FGF expression suggest that these growth factors are involved in the integrit...

ZAM is an env-containing member of the gypsy family of retrotransposons that represents a possible retrovirus of invertebrates. In this paper, we traced ZAM mobilization to get information about a potential path a retroelement may take to reach the germ line of its host. In situ hybridization on whole-mount tissues and immunocytochemistry analyses with antibodies raised against ZAM Gag and Env proteins have shown that all components necessary to assemble ZAM viral particles, i.e., ZAM full-length RNAs and Gag and Env polypeptides, are coexpressed in a small set of follicle cells surrounding the oocyte. By electron microscopy, we have shown that ZAM viral particles are indeed detected in this somatic lineage of cells, which they leave and enter the closely apposed oocyte. Our data provide evidence that the vesicular traffic and yolk granules in the process of vitellogenesis play an important role in ZAM transfer to the oocyte. Our data support the possibility that vitellogenin transfer to the oocyte may help a retroelement pass to the germ line with no need of its envelope product.ZAM is a 8.4-kb retroelement that resides within the genome of Drosophila melanogaster (11). On the basis of sequence similarity and gene organization, ZAM is a member of a group of retrotransposons that bears a striking resemblance to the vertebrate retroviruses. These elements are flanked by long terminal repeats (LTRs) that direct the transcription of fulllength RNAs representing potential templates for reverse transcription during mobilization. The LTRs flank three open reading frames (ORFs) analogous in position and coding potential to the retroviral gag, pol, and env genes ( Fig. 1). Among the diverse classes of eukaryotic retrotransposons, the presence of a third env-like ORF (ORF3) is unique to ZAM and a small group of other members of this family, including gypsy, 297, 17.6, Idefix, and nomad in D. melanogaster (3,8,14,19,26), tom in Drosophila ananassae (25), Osvaldo in Drosophila buzzatii (15), TED in the lepidopteran Trichoplusia ni (5), and Yoyo in the medfly Ceratitis capitata (28). An envelope protein expressed in vivo has been identified for only three of these elements (gypsy, tom, and TED) (16,21,24,25), and only one of them, gypsy, has been shown to date to have infectious properties (9,22). Although retroviral Env proteins are known to be involved in viral infectivity through host cell receptor recognition and fusion of viral and cellular membranes, the role of the Env glycoproteins encoded by these elements is still unclear since no budding has ever been visualized for any of them.ZAM was first identified as a spontaneous insertion at the white locus, giving rise to the w IR6RevI allele in a line of D. melanogaster subsequently called RevI (11). This mutation occurred in the course of a massive amplification of ZAM elements in this line due to their mobilization, which remains active in this stock of flies (3). The existence of RevI and its parental line, w IR6 , which displays a low copy number of stable ZAM elemen...

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.

We describe a novel transposition system in a line of Drosophila melanogaster called RevI in which two retroelements are mobilized. These elements are the retroelement ZAM, recently described in the literature, and a novel element designated Idefix. Like ZAM, Idefix displays 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. In situ hybridization and Southern analyses performed on the RevI genome revealed the presence of some 20 copies of ZAM and Idefix, whereas ZAM is absent and Idefix is present in only four copies on the chromosomal arms of the original parental line. From RevI, a series of mutations affecting eye coloration has been recovered. The genetic and molecular analyses of these mutations have shown that most of them affected the white locus through three rounds of mutational events. The first mutational event was previously shown to be caused by a ZAM insertion 3 kb upstream of the transcription start site of white. It confers a red-brick phenotype to the orange eye coloration of the parental line. The second event results from the insertion of an Idefix copy 1.7 kb upstream of the transcription start site of the white gene, which modifies the red-brick phenotype to orange. This second mutational event was recovered as a recurrent specific mutation in 11 independent individuals. The third event results from an additional Idefix located 1.7 kb upstream of white that is responsible for the full reversion of the orange phenotype to red-brick. The fact that such mutations due to recurrent appearances of both ZAM and Idefix at the white locus result in such a variety of phenotypes brings to light a new molecular system in which the interference of mobile elements with the correct expression of the host gene can be tested.

BackgroundIn the Drosophila germ line, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous transposable elements. This RNA silencing involves small RNAs of 26-30 nucleotides that are mainly produced from the antisense strand and function through the Piwi protein. Piwi belongs to the subclass of the Argonaute family of RNA interference effector proteins, which are expressed in the germline and in surrounding somatic tissues of the reproductive apparatus. In addition to this germ-line expression, Piwi has also been implicated in diverse functions in somatic cells.Principal FindingsHere, we show that two LTR retrotransposons from Drosophila melanogaster, ZAM and Idefix, are silenced by an RNA silencing pathway that has characteristics of the rasiRNA pathway and that specifically recognizes and destroys the sense-strand RNAs of the retrotransposons. This silencing depends on Piwi in the follicle cells surrounding the oocyte. Interestingly, this silencing is active in all the somatic tissues examined from embryos to adult flies. In these somatic cells, while the silencing still involves the strict recognition of sense-strand transcripts, it displays the marked difference of being independent of the Piwi protein. Finally, we present evidence that in all the tissues examined, the repression is controlled by the heterochromatic COM locus.ConclusionOur data shed further light on the silencing mechanism that acts to target Drosophila LTR retrotransposons in somatic cells throughout fly development. They demonstrate that different RNA silencing pathways are involved in ovarian versus other somatic tissues, since Piwi is necessary for silencing in the former tissues but is dispensable in the latter. They further demonstrate that these pathways are controlled by the heterochromatic COM locus which ensures the overall protection of Drosophila against the detrimental effects of random retrotransposon mobilization.

NucleusJ 1.0, an ImageJ plugin, has been shown to be a useful tool to analyse nuclear morphology and chromatin organisation in plant and animal cells. However, technological improvements of confocal microscopy have speeded-up image acquisition, highlighting the bottleneck in 3D image analysis caused by manual steps in NucleusJ 1.0 and limiting its use for big data analysis. NucleusJ 2.0 is a new release of NucleusJ, in which image processing is achieved more quickly using a command-line user interface. Starting with large collection of 3D nuclei, segmentation can be performed by the previously developed Otsu-modified method or by a new 3D gift-wrapping method, taking better account of nuclear indentations and unstained nucleoli. These two complementary methods based on threshold and edgebased detection, are compared for their accuracy by using three types of datasets-digitised spheres, microspheres and plant nuclei-available to the community through an open source repository accessible at https://www.brookes.ac.uk/indepth/images/. A discrete geometric method was introduced to improve the surface area calculation, a key parameter when studying nuclear morphology, replacing an ImageJ default tool by a new one that includes pixel context information. Finally, NucleusJ 2.0 was evaluated using original plant genetic material, in which nuclear morphology and chromatin organisation are strongly affected and by assessing its efficiency on nuclei stained with DNA dyes or after 3D-DNA Fluorescence in situ hybridisation. With these improvements, NucleusJ 2.0 permits the generation of large user-curated datasets that will be useful for software benchmarking or to train convolution neural networks.