Molecular Biology Reports

, Volume 38, Issue 1, pp 355–358

Localized expression pattern of miR-184 in Drosophila

Authors

  • Ping Li
    • Key Laboratory of Developmental Genes and Human Disease, Ministry of EducationSoutheast University
    • Institute of Life ScienceSoutheast University
  • Jianjian Peng
    • Key Laboratory of Developmental Genes and Human Disease, Ministry of EducationSoutheast University
    • Institute of Life ScienceSoutheast University
  • Jiangbo Hu
    • Key Laboratory of Developmental Genes and Human Disease, Ministry of EducationSoutheast University
    • Institute of Life ScienceSoutheast University
  • Zhongxin Xu
    • Key Laboratory of Developmental Genes and Human Disease, Ministry of EducationSoutheast University
    • Institute of Life ScienceSoutheast University
  • Wei Xie
    • Key Laboratory of Developmental Genes and Human Disease, Ministry of EducationSoutheast University
    • Institute of Life ScienceSoutheast University
    • Key Laboratory of Developmental Genes and Human Disease, Ministry of EducationSoutheast University
    • Institute of Life ScienceSoutheast University
Article

DOI: 10.1007/s11033-010-0115-1

Cite this article as:
Li, P., Peng, J., Hu, J. et al. Mol Biol Rep (2011) 38: 355. doi:10.1007/s11033-010-0115-1

Abstract

MicroRNAs (miRNAs) are a kind of endogenous non-coding small RNAs whose specific functions in animals are generally important. Although functions of some miRNAs have been identified, the role of miR-184 remains unknown. Here, we determined the temporal and spatial expression pattern of miR-184 during the different development stages and tissues in Drosophila. Strikingly, miR-184 is expressed ubiquitously in Drosophila embryos, larvae and adults, its expression pattern shows a dynamic changes during the development of embryo, especially in the central nervous system. This expression profile suggests that miR-184 may act important function in Drosophila development.

Keywords

miR-184Expression patternDrosophila

MicroRNAs (miRNAs) are 21–24 nt non-coding RNAs that form duplexes with target mRNA transcripts and induce transcript cleavage and/or inhibit productive translation. Since the identified of the first miRNAs lin-4 in Caenorhabditis elegans in 1993 [1], it has been reported about 175 miRNAs in worms, 157 miRNAs in flies, 579 miRNAs in mouse and approximately 721 miRNAs in human (http://www.mirbase.org). miRNAs are universally present in animals, plants and viruses that play an important role in the development, proliferation, differentiation, apoptosis of organisms and cancers [25].

Most miRNAs display a tissue- and/or developmental-specific expression pattern, which suggests highly specific and diverse roles for miRNAs. For example, miR-1 is found exclusively in muscles, where it regulates cardiac differentiation in Drosophila [6], miR-9a is specifically expressed in epithelial cells and proved to controls the generation of sensory organs [7], miR-278 is specifically expressed in adipose cells, where it regulates genes involved in insulin secretion [8].

miR-184 is a single copy gene and evolutionarily conserved at the nucleotide level from flies to humans. It is expressed in the mesoderm in situ hybridization in Drosophila [9]. In Zebrafish, it is expressed in lens, hatching gland and epidermis by Northern blots using LNA (locked-nucleic acid)-modified DNA oligonucleotide probes [10]. An analysis of the primary transcript of miR-184 (pri-mir-184) in several mouse tissues revealed specific expression in brain and testis, its expression that repressed by the binding of MeCP2 to its promoter, is upregulated by the release of MeCP2 after depolarization, this give a clue to link between the miRNAs and DNA methylation pathways [11]. Over expression of miR-184 in neuroblastoma cell lines results in massive apoptosis [12]. A recent paper discovers miR-184 has multiple roles in Drosophila female germline development [13]. Together, these findings indicate that miR-184 can play essential roles in development. However, the temporal and spatial expression pattern of miR-184 is still being debated.

In this paper, we examined the expression pattern of miR-184 throughout the life cycle and different discs of Drosophila by in situ hybridization and a modified ribonuclease protection assay (RPA) method. We give some clues to the miR-184 studies.

Materials and methods

RT-PCR

RNA isolation and reverse transcription (RT-PCR) of whole samples were performed according to standard protocols. An oligo dT18 primer was used for the RT step and the gene-specific primer pairs were used for the PCR. Primer sequences are 5′-TCGGATACGGATACGGATTCCTG-3′ and 5′-ATGCACATGTTGGCAGACAGC-3′. Ten percent of the RT product served as the template for the PCR, which was kept in the linear range of amplification. The products were separated on 2% agrose gel.

In situ hybridization

A 500 bp DNA fragment of miR-184 precursor were amplified from Drosophila genomic DNA by PCR, Primers are 5′-TGCGCACGTTCAATTTGCAA-3′ and 5′-ATGAGTTGGCAGACAGCAGC-3′. The 500 bp fragment was labeled with digoxigenin-UTP (Roche Company) by using the T7 transcriptase in vitro according to the manufacturer’s instructions and used as probe.

Embryos were collected up to 17 h, fixed and prepared by using standard protocols. Embryos were prehybridized for 1 h and then hybridized in 0.2 ng/μl Dig-probe for 12–18 h at 55°C and incubated with anti-Dig Fab alkaline phosphatase coupled antibody overnight at 4°C at a dilution 1:2000. The pri-mir-184 was detected by AP substrate solution (NBT & BCIP).

Ribonuclease protection assay

The ribonuclease protection assay was carried out mainly according to the mirVana miRNA Detection Kit Instructions (Ambion Company) with the following modifications. The miR-184 was detected with a purified biotin-labeled oligo probe by using the T7 RNA polymerase in vitro according to the manufacturer’s instructions, the 5S rRNA was used as control. The probe sequence is 5′-GAGACAGGGCCCUU-AUCAGUUCUCCGUCCA-3′, which contains a 22 nt sequence (GCCCUUAUCA-GUUCUCCGUCCA) that is the complement of the mature miR-184 sequence and 8 nt (GAGACAGG) nonspecific sequence that will not hybridize with the target RNA. Hybridization was performed at 42°C. Un-hybridized probe and RNAs were digested by RNase A/RNase T1. The RNases activity was inactivated by RNase Inactivation/PPT solution and protected RNA fragments were precipitated using ethanol. RNAs were separated on 15% denaturing polyacrylamide gel, transferred to Hybond nylon membrane (Biodyne B) and united with nylon membrane by 1200J UV crossing. miR-184 was probed with Streptividin-HRP (Genscript Company) at a dilution 1:20000. Cross reacting bands were visualized using the ECL detection kit (Thermo Company). The sequence in the probe that does not hybridize to the target is longer than the protected fragment so that an obvious difference in size is seen between the full-length undigested probe and the protected fragment after RNase digestion. The shift in size from full-length to a smaller protected fragment helps to validate that RNA from the sample is protecting the probe and is not an artifact.

Results and discussion

miR-184 was originally identified by expression cloning from the small RNA fraction of Drosophila embryos, but is evolutionarily conserved at the nucleotide level from insects to humans [14], the mature nucleotide sequences in human resembles in mouse completely, only one nucleotide is different between human and fly (Fig. 1a). miR-184 gene is located on the right arm of the second chromosome 50A1, 30 kb to the left gene CG17047 and 24 kb to the right gene CG17048 (Fig. 1b). To focus future miR-184 function studies, we determined the spatial and temporal expression patterns of miR-184 in Drosophila development.
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-010-0115-1/MediaObjects/11033_2010_115_Fig1_HTML.gif
Fig. 1

Drosophila miR-184 locus and physical map, mir-184 gene is located on the right arm of the second chromosome 50A1, 30 kb to the left gene CG17047 and 24 kb to the right gene CG17048, mature miR-184 is evolutionarily conserved at the nucleotide level from insects to humans. a Nucleotide sequence alignment of miR-184 among different species. b Organization of the mir-184 locus in the Drosophila genome

First, we determined the temporal expression of miR-184 during Drosophila development, the primary transcript of miR-184 (pri-miR-184) was identified by RT-PCR analysis using the primer around the mature miR-184 coding sequence. miR-184 can be detected during different embryo stages (Fig. 2a), larvae and adult (Fig. 2c), miRNA-184 became visible at stages1-4, and become increasingly enriched at stages 12–13. In larvae and adults, miR-184 remained expressed, and showed strong expression at L3. An analysis of the mature miR-184 by modified ribonuclease protection assay also gave similar results (Fig. 2b–d).
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-010-0115-1/MediaObjects/11033_2010_115_Fig2_HTML.gif
Fig. 2

Expression patterns of miR-184 at different stages in Drosophila. miR-184 is expressed in a highly dynamic pattern that arising during the fly development and its fastigium expression is at stages 12–13 and L3. apri-mir-184 level detected throughout embryogenesis in wild-type by RT-PCR, including stage 1–4, stage 5, stage 6–7, stage 8–11, stage 12–13, stage 14–16, stage 17. b Mature miR-184 level detected throughout embryogenesis in wild-type by modified PRA. cpri-mir-184 level detected in wild-type larvae and adult by RT-PCR, including first instar larvae (L1), second instar larvae (L2), third instar larvae (L3), pupa (Pupa), male fly (Male) and female fly (Female). d Mature miR-184 level detected in wild-type larvae and adult by modified PRA

In addition, we determined the expression of miR-184 in developing imaginal discs of larvae. An analysis of the primary transcript of miR-184 (pri-mir-184) in several discs revealed ubiquitous expression in head, eye and wing discs (Fig. 3a). An analysis of the mature miR-184 by modified ribonuclease protection assay also gave similar results (Fig. 3b).
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-010-0115-1/MediaObjects/11033_2010_115_Fig3_HTML.gif
Fig. 3

Expression patterns of miR-184 in different tissues in Drosophila, miR-184 is expression generally, relative high at head disc and eye disc. apri-mir-184 level detected in head disc (head), eye disc (eye), wing disc (wing) by RT-PCR. b Mature miR-184 level detected in head disc (head), eye disc (eye), wing disc (wing) by modified PRA

Figure 4 summarizes the miR-184 expression pattern as revealed by in situ hybridization with RNA probes to whole mount preparations of embryos. miR-184 shows strong expression in a highly dynamic pattern throughout embryogenesis and present especially in the brain and ventral nerve cord. miR-184 transcripts are present at high levels in eggs and early embryos up to the gastrulation stage (Fig. 4A). Discernible expression is detected during gastrulation, covering the area of the differentiating neuroectoderm of stage 7 embryos (Fig. 4B, C). At late stage 14, miR-184 transcripts are highly enriched, mainly expressed in the central nervous system (CNS) and brain of the embryo (Fig. 4D–I).
https://static-content.springer.com/image/art%3A10.1007%2Fs11033-010-0115-1/MediaObjects/11033_2010_115_Fig4_HTML.jpg
Fig. 4

Transcript pattern of the pri-mir-184 visualized throughout embryogenesis by in situ hybridization. miR-184 is expressed in a highly dynamic pattern throughout embryogenesis, especially in CNS. The whole mount embryos stages are including stage 1 (A), stage 7 (B, C), stage 14 (D, E), stage 15 (F, G) and stage 17 (H, I). Wild-type embryos are shown in lateral (B, D, F, H) and ventral view (C, E, G, I)

In conclusion, we here describe the temporal and spatial expression pattern of miR-184 in Drosophila development, miR-184 expression in the embryo is neurospecific whereas in the larvae, transcripts are also found in several discs (e.g. head, eye and wing discs). This expression profile suggests that miR-184 may play crucial roles in Drosophila development, including tissue fate establishment, differentiation or the maintenance of tissue identity.

Acknowledgements

We thank the Bloomington stocks center for fly stocks, this work was supported by NSFC (30670444).

Copyright information

© Springer Science+Business Media B.V. 2010