Project description:Single microRNAs are usually associated with hundreds of putative target genes that can influence multiple phenotypic traits in Drosophila, ranging from development to behaviour. We investigated the function of Drosophila miR-210 in circadian behaviour by misexpressing it within circadian clock cells. Manipulation of miR-210 expression levels in the PDF (pigment dispersing factor) positive neurons affected the phase of locomotor activity, under both light-dark conditions and constant darkness. PER cyclical expression was not affected in clock neurons, however, when miR-210 was up-regulated, a dramatic alteration in the morphology of PDF ventral lateral neuron (LNv) arborisations was observed. The effect of miR-210 in shaping neuronal projections was confirmed in vitro, using a Drosophila neuronal cell line. A transcriptomic analysis revealed that miR-210 overexpression affects the expression of several genes belonging to pathways related to circadian processes, neuronal development, GTPases signal transduction and photoreception. Collectively, these data reveal the role of miR-210 in modulating circadian outputs in flies and guiding/remodelling PDF positive LNv arborisations and indicate that miR-210 may have pleiotropic effects on the clock, light perception and neuronal development.
Project description:MicroRNAs (miRNAs) are a novel class of small RNAs which act as modulators of gene expression either by inhibiting the translation or by inducing the degradation of their target mRNAs. Several studies suggest a role for miRNAs as regulators of the circadian clock in mammals and Drosophila. Based on computational predictions of target mRNAs of clock (or clock related) genes, we have selected the miR-210 as a putative regulator of the period clock gene. We demonstrated that flies over-expressing this miRNA in the canonical clock neurons show an impaired locomotor activity pattern in both light-dark (LD) and constant darkness conditions (DD). Moreover, the projections of the Pigment Dispersing Factor (PDF)-expressing clock neurons in the optic lobe are abnormal showing peculiar “star” shaped body cells. The microarray analysis performed in the adult fly brain, revealed that this miRNA is affecting indirectly the expression of some circadian genes (ie: pdf, npf, cry, tim, pdp1) but not the period gene, and directly genes like echinus and RhoGAp92B. The in-vivo down regulation of echinus indeed can be associated with a severe impairment of the locomotor activity of flies, while the GTPase RhoGAP92B, important for the regulation of the cellular shape of neurons, could be involved in the morphological development of the PDF-expressing neurons.
Project description:MicroRNAs (miRNAs) are a novel class of small RNAs which act as modulators of gene expression either by inhibiting the translation or by inducing the degradation of their target mRNAs. Several studies suggest a role for miRNAs as regulators of the circadian clock in mammals and Drosophila. Based on computational predictions of target mRNAs of clock (or clock related) genes, we have selected the miR-210 as a putative regulator of the period clock gene. We demonstrated that flies over-expressing this miRNA in the canonical clock neurons show an impaired locomotor activity pattern in both light-dark (LD) and constant darkness conditions (DD). Moreover, the projections of the Pigment Dispersing Factor (PDF)-expressing clock neurons in the optic lobe are abnormal showing peculiar “star” shaped body cells. The microarray analysis performed in the adult fly brain, revealed that this miRNA is affecting indirectly the expression of some circadian genes (ie: pdf, npf, cry, tim, pdp1) but not the period gene, and directly genes like echinus and RhoGAp92B. The in-vivo down regulation of echinus indeed can be associated with a severe impairment of the locomotor activity of flies, while the GTPase RhoGAP92B, important for the regulation of the cellular shape of neurons, could be involved in the morphological development of the PDF-expressing neurons.
Project description:MicroRNAs (miRNAs) are small non-protein-coding RNAs that are incorporated into the RNA-induced silencing complex (RISC) and inhibit gene expression by regulating the stability and/or the translational efficiency of target mRNAs. Previously, we demonstrated that miR-210 is a key player of endothelial cell (EC) response to hypoxia, modulating EC survival, migration and ability to form capillary like-structures. Moreover, the receptor tyrosine kinase ligand Ephrin-A3 was identified as one functionally relevant target. Since each miRNA regulates hundreds of mRNAs, different approaches were combined to identify new miR-210 targets: a Using target prediction software, 32 new miR-210 potential targets were identified. b The proteomic profiling of miR-210 over-expressing ECs identified 11 proteins that were specifically inhibited by miR-210, either directly or indirectly. c Affymetrix based gene expression profiles identified 51 genes that were both down-modulated by miR-210 over-expression and de-repressed when miR-210 was blocked. Surprisingly, only few genes identified either by proteomics or transcriptomics were recognized as miR-210 targets by target prediction algorithms. However, a low-stringency pairing research revealed enrichment for miR-210 putative binding sites, raising the possibility that these genes were targeted via non-canonical recognition sequences. To clarify this issue, miR-210-loaded RISC was purified by immuno-precipitation along with its mRNA targets. The presence of Ephrin-A3 mRNA in the complex validated this approach. We found that 32 potential targets were indeed enriched in miR-210-loaded RISC, and thus can be considered as genuine miR-210 targets. In keeping with this conclusion, we were able to further validate a sub-set of them by 3âUTR-reporter assays. Gene ontology analysis of the targets confirmed the known miR-210 activity in differentiation and cell cycle regulation, highlighting new functions such as involvement in RNA processing, DNA binding, development, membrane trafficking and amino acid catabolism. In conclusion, we validated a multidisciplinary approach for miRNAs target identification and indicated novel molecular mechanisms underpinning miR-210 role in EC response to hypoxia. Experiment Overall Design: Gene expression modifications induced by both miR-210 over-expression and blockade were evaluated. In order to identify new direct and indirect miR-210 targets, transcripts repressed by miR-210 over-expression and up-regulated by miR-210 inhibition (and vice versa) were selected.
Project description:we reported single-cell gene expression of CD4+ T cells from the visceral adipose tissue of from male Foxp3-Cre.YFP bmal1WT or Foxp3-Cre.YFP bmal1flox/flox mice, and from spleen from bmal1WT mice, at ZT0 or ZT12. We found that VAT Tregs could be subdivided into five subtypes: p1 ST2+, p2 ST2+, Tbet+, IL18r+ and resting clusters. We found diurnal variation within the ST2+ subgroups, where the more activated p1 ST2+ Tregs were more represented at ZT0 in WT VAT Tregs. Such variations were not observed in splenic Tregs. In contrast, bmal1KO VAT Tregs were were enriched in the ST2+ Tregs, and had a constitutively high proportion of the p1 ST2+ subtype at ZT0 and ZT12.
Project description:RNAseq transcriptional profiling of Drosophila brains from wildtype, and period loss-of-function animals with time points taken over two days. 2 days of brain collection, time points at ZT0, ZT6, ZT12, and ZT18; wildtype and per0 flies. 10-12 brains per time point.
Project description:MicroRNAs (miRNAs) are small non-protein-coding RNAs that are incorporated into the RNA-induced silencing complex (RISC) and inhibit gene expression by regulating the stability and/or the translational efficiency of target mRNAs. miR-210 can be considered a master miRNA of hypoxic response and is currently regarded as a promising novel non-invasive tumor hypoxia marker. The targets identified to date indicate that miR-210 plays a role in cell cycle regulation, differentiation, mitochondrial metabolism repression, DNA repair and apoptosis. In order to identify miRNAs sub-sequentely modulated by miR-210, miRNA expression profiles of human umbilical vein endothelial cells (HUVEC) over-expressing miR-210 were generated, allowing the identification of miRNAs modulated upon miR-210 up-regulation.