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International audience; MicroRNAs are small non-coding RNAs that are now recognised as key regulators of gene expression in eukaryotes. Over the past few years, hundreds of miRNAs have been identified from various organisms including vertebrates, nematodes, insects and plants. A high level of conservation of some miRNAs from animals to plants underlines their crucial role in eukaryotes. Although biogenesis and mode of action of miRNAs are now quite well understood, their numerous and specific regulatory functions remain to be unravelled. In this review, we summarise the current knowledge on miRNAs in insects, which was mainly acquired through the study of the fruit fly, Drosophila melanogaster.

Supplemental material: http://rnajournal.cshlp.org/content/suppl/2011/12/01/rna.028878.111.DC1.html; Over the last years, the microRNA (miRNA) pathway has emerged as a key component of the regulatory network of pluripotency. Although clearly distinct states of pluripotency have been described in vivo and ex vivo, differences in miRNA expression profiles associated with the developmental modulation of pluripotency have not been extensively studied so far. Here, we performed deep sequencing to profile miRNA expression in naive (embryonic stem cell [ESC]) and primed (epiblast stem cell [EpiSC]) pluripotent stem cells derived from mouse embryos of identical genetic background. We developed a graphical representation method allowing the rapid identification of miRNAs with an atypical profile including mirtrons, a small nucleolar RNA (snoRNA)-derived miRNA, and miRNAs whose biogenesis may differ between ESC and EpiSC. Comparison of mature miRNA profiles revealed that ESCs and EpiSCs exhibit very different miRNA signatures with one third of miRNAs being differentially expressed between the two cell types. Notably, differential expression of several clusters, including miR290-295, miR17-92, miR302/367, and a large repetitive cluster on chromosome 2, was observed. Our analysis also showed that differentiation priming of EpiSC compared to ESC is evidenced by changes in miRNA expression. These dynamic changes in miRNAs signature are likely to reflect both redundant and specific roles of miRNAs in the fine-tuning of pluripotency during development.

Background The ventral midbrain contains a diverse array of neurons, including dopaminergic neurons of the ventral tegmental area (VTA) and substantia nigra (SN) and neurons of the red nucleus (RN). Dopaminergic and RN neurons have been shown to arise from ventral mesencephalic precursors that express Sonic Hedgehog (Shh). However, Shh expression, which is initially confined to the mesencephalic ventral midline, expands laterally and is then downregulated in the ventral midline. In contrast, expression of the Hedgehog target gene Gli1 initiates in the ventral midline prior to Shh expression, but after the onset of Shh expression it is expressed in precursors lateral to Shh-positive cells. Given these dynamic gene expression patterns, Shh and Gli1 expression could delineate different progenitor populations at distinct embryonic time points. Results We employed genetic inducible fate mapping (GIFM) to investigate whether precursors that express Shh (Shh-GIFM) or transduce Shh signaling (Gli1-GIFM) at different time points give rise to different ventral midbrain cell types. We find that precursors restricted to the ventral midline are labeled at embryonic day (E)7.5 with Gli1-GIFM, and with Shh-GIFM at E8.5. These precursors give rise to all subtypes of midbrain dopaminergic neurons and the anterior RN. A broader domain of progenitors that includes the ventral midline is marked with Gli1-GIFM at E8.5 and with Shh-GIFM at E9.5; these fate-mapped cells also contribute to all midbrain dopaminergic subtypes and to the entire RN. In contrast, a lateral progenitor domain that is labeled with Gli1-GIFM at E9.5 and with Shh-GIFM at E11.5 has a markedly reduced potential to give rise to the RN and to SN dopaminergic neurons, and preferentially gives rise to the ventral-medial VTA. In addition, cells derived from Shh- and Gli1-expressing progenitors located outside of the ventral midline give rise to astrocytes. Conclusions We define a ventral midbrain precursor map based on the timing of Gli1 and Shh expression, and suggest that the diversity of midbrain dopaminergic neurons is at least partially determined during their precursor stage when their medial-lateral position, differential gene expression and the time when they leave the ventricular zone influence their fate decisions. Background The ventral mesencephalic progenitor domain generates a diverse array of distinct neuronal cell types, including neurons of the red nucleus (RN), motoneurons of the oculomotor nucleus and midbrain dopaminergic (DA) neurons. DA neurons are further organized into anatomically and functionally distinct subclasses [1]. The substantia nigra (SN), located in the lateral-ventral midbrain, projects to the dorsal-lateral striatum and is involved in the regulation of motor behaviors. The ventral tegmental area (VTA), located more medially, projects to corticolimbic targets and is important for motivational states. The retrorubral field is located posterior to the SN and projects to striatal, limbic and cortical areas. The functional diversity of these different regions becomes apparent in disease states: in Parkinson's disease, SN neurons, but not VTA neurons, degenerate, resulting in severe motor deficits. In contrast, abnormalities in the mesocorticolimbic system have been implicated in addiction, schizophrenia and attention deficit disorder [2–4]. While it is well established that the functional diversity of ventral midbrain neurons and DA subclasses is based on their distinct efferent and afferent connections and their distinct molecular make-up and physiology, it remains unclear when and how these distinct neuronal (sub)classes are established during development. All midbrain DA neurons appear to arise from ventral mesencephalic floor plate progenitors that express Sonic Hedgehog (Shh) [5–8]. A recent paper utilizing genetic inducible fate mapping (GIFM) [9] suggested that Shh expression between embryonic day (E)7.5 and E12.5 sequentially marks three spatially distinct ventral mesencephalic progenitor domains that give rise to different neurons. However, the distribution of fate-mapped cells was only assessed qualitatively at embryonic stages, and a potential contribution to glia was not determined. Gli1, a zinc finger transcription factor in the Shh signaling pathway, is only transcribed in cells that receive high levels of Hedgehog signaling (and are close to the source of Hedgehog) [10, 11]; therefore, its expression can be used as a readout for cells that are exposed to high levels of Shh signaling [12]. Shh-expressing cells, including the floor plate cells themselves, do not respond to Shh signaling as measured by the expression of Gli1 [11–13]. It is therefore necessary to understand the exact timing of Shh responses and Shh expression in ventral midbrain precursors to gain a better insight into the role of Shh signaling in specification of ventral midbrain neurons. To establish a precise precursor map of the ventral mesencephalon, we assessed the fate of Gli1-expressing (Shh-responding) and Shh-expressing progenitors with GIFM in a quantitative manner at embryonic and postnatal stages. We show that Gli1 expression precedes Shh expression by about a day and demonstrate that ventral midbrain precursors that give rise to DA neurons respond to Shh signaling between E7.5 and E9.5 and express Shh between E8.5 and E11.5. Progenitors in the ventral midline that are labeled with Gli1-GIFM at E7.5 and with Shh-GIFM at E8.0 to E8.5 contribute to midbrain DA neurons and the anterior RN. Progenitors in a broader domain are marked with Gli1-GIFM at E8.5 and Shh-GIFM at E9.5 to E10.5 and show a strong contribution to all subsets of DA neurons and to RN neurons. Precursors adjacent to the ventral midline that are fate-mapped with Gli1-GIFM at E9.5 and Shh-GIFM at E11.5 maintain the potential to develop into DA neurons of the ventral-medial VTA. However, they contribute few cells to DA neurons in the SN and to RN neurons. In addition, precursors labeled with Gli1-GIFM at E8.5 to E9.5 give rise to other ventral midbrain neurons, including neurons in the oculomotor nucleus and the non-DA neurons in the SN reticularis, consistent with a broad medial-lateral distribution of Gli1-expressing precursors. Finally, we observe that Shh- and Gli1-expressing progenitors, with the exception of progenitors in the ventral midline, develop into ventral midbrain astrocytes.

Contains fulltext : 80137.pdf (Publisher’s version ) (Closed access) Multicellular organisms evolved sophisticated defence systems to confer protection against pathogens. An important characteristic of these immune systems is their ability to act both locally at the site of infection and at distal uninfected locations. In insects, such as Drosophila melanogaster, RNA interference (RNAi) mediates antiviral immunity. However, the antiviral RNAi defence in flies seems to be a local, cell-autonomous process, as flies are thought to be unable to generate a systemic RNAi response. Here we show that a recently defined double-stranded RNA (dsRNA) uptake pathway is essential for effective antiviral RNAi immunity in adult flies. Mutant flies defective in this dsRNA uptake pathway were hypersensitive to infection with Drosophila C virus and Sindbis virus. Mortality in dsRNA-uptake-defective flies was accompanied by 100-to 10(5)-fold increases in viral titres and higher levels of viral RNA. Furthermore, inoculating naked dsRNA into flies elicited a sequence-specific antiviral immune response that required an intact dsRNA uptake pathway. These findings suggest that spread of dsRNA to uninfected sites is essential for effective antiviral immunity. Notably, infection with green fluorescent protein (GFP)-tagged Sindbis virus suppressed expression of host-encoded GFP at a distal site. Thus, similar to protein-based immunity in vertebrates, the antiviral RNAi response in flies also relies on the systemic spread of a virus-specific immunity signal.

International audience; Although it has been well established that histone acetyltransferases (HATs) are involved in the modulation of chromatin structure and gene transcription, there is only little information on their developmental role in higher organisms. Gcn5 was the first transcription factor with HAT activity identified in eukaryotes. Here we report the isolation and characterization of Drosophila melanogaster dGcn5 mutants. Null dGcn5 alleles block the onset of both oogenesis and metamorphosis, while hypomorphic dGcn5 alleles impair the formation of adult appendages and cuticle. Strikingly, the dramatic loss of acetylation of the K9 and K14 lysine residues of histone H3 in dGcn5 mutants has no noticeable effect on larval tissues. In contrast, strong cell proliferation defects in imaginal tissues are observed. In vivo complementation experiments revealed that dGcn5 integrates specific functions in addition to chromosome binding and acetylation. Surprisingly, a dGcn5 variant protein with a deletion of the bromodomain, which has been shown to recognize acetylated histones, appears to be fully functional. Our results establish dGcn5 as a major histone H3 acetylase in Drosophila which plays a key role in the control of specific morphogenetic cascades during developmental transitions.

International audience; Defects in miRNA biogenesis or activity are associated to development abnormalities and diseases. In Drosophila, miRNAs are predominantly loaded in Argonaute-1, which they guide for silencing of target RNAs. The miRNA pathway overlaps the RNAi pathway in this organism, as miRNAs may also associate with Argonaute-2, the mediator of RNAi. We set up a gene construct in which a single inducible promoter directs the expression of the GFP protein as well as two miRNAs perfectly matching the GFP sequences. We show that self-silencing of the resulting automiG gene requires Drosha, Pasha, Dicer-1, Dicer-2 and Argonaute-2 loaded with the anti-GFP miRNAs. In contrast, self-silencing of the automiG gene does not involve Argonaute-1. Thus, automiG reports in vivo for both miRNA biogenesis and Ago-2 mediated silencing, providing a powerful biosensor to identify situations where miRNA or siRNA pathways are impaired. As a proof of concept, we used automiG as a biosensor to screen a chemical library and identified 29 molecules that strongly inhibit miRNA silencing, out of which 5 also inhibit RNAi triggered by long double-stranded RNA. Finally, the automiG sensor is also self-silenced by the anti-GFP miRNAs in HeLa cells and might be easily used to identify factors involved in miRNA biogenesis and silencing guided by perfect target complementarity in mammals.