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Gene expression analysis of miR-210 over-expressing flies at ZT12.

ABSTRACT: 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.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE77233 | GEO | 2018/11/13

REPOSITORIES: GEO

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Project description:To compare circadian gene expression within highly discrete neuronal populations, we separately purified and characterized two adjacent but distinct groups of Drosophila adult circadian neurons: the 8 small and 10 large PDF (pigment-dispersing factor)-expressing ventral lateral neurons (s-LNvs and l-LNvs, respectively). The s-LNvs are the principal circadian pacemaker cells, whereas recent evidence indicates that the l-LNvs are involved in sleep and light-mediated arousal. Although half of the l-LNv-enriched mRNA population including core clock mRNAs is shared between the l-LNvs and s-LNvs, the other half is l-LNv- and s-LNv specific. The distribution of four specific mRNAs is consistent with prior characterization of the four encoded proteins and therefore indicates successful purification of the two neuronal types. Moreover, an octopamine receptor mRNA is selectively enriched in l-LNvs, and only these neurons respond to in vitro application of octopamine. Dissection and purification of l-LNvs from flies collected at different times indicate that these neurons contain cycling clock mRNAs with higher circadian amplitudes as well as at least a 10-fold higher fraction of oscillating mRNAs than all previous analyses of head RNA. Many of these cycling l-LNv mRNAs are well-expressed but do not cycle or cycle much less well elsewhere in heads. The results suggest that RNA cycling is much more prominent in circadian neurons than elsewhere in heads and may be particularly important for the functioning of these neurons. Gene expression was profiled in purified large PDF neurons across 4 time-points and small PDF neurons at two time-points under an LD cycle. Circadian neurons (PDF large and small cells), general neurons (ELAV) and per01:large PDF cells were labeled by GFP using specfic drivers. Expression of these cells types were profiled after manual cell sorting of GFP-positive cells at different time-points under a light/dark (LD) cycle. The time-points included ZT0, ZT6, ZT12, and ZT18 in the case of large PDF cells, and ZT0 and ZT12 in the case of small PDF cells, ELAV cells and per01:large PDF cells. 3 biological replicates were collected for the large PDF cells and ELAV cells at ZT0 and ZT12. 2 replicates were collected for large PDF cells at ZT6 and ZT18, small PDF cells, and per01:large PDF cells.

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Gene expression analysis of miR-210 over-expressing flies at ZT12.

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