InteractiveFly: GeneBrief

mir-184 stem loop: Biological Overview | References

BIOLOGICAL OVERVIEW

The regulation of gene expression during early development is very complex. In nonplacental organisms, the mother initiates and controls much of this process by placing mRNA transcripts in well-defined concentrations and locations within the developing egg. In many instances, these maternal 'determinants' serve as morphogens -- their absolute and relative concentrations are therefore crucial and under elaborate regulation, which includes mechanisms for transporting and localizing transcripts and tight control of their translation. During the midblastula transition, many of the maternal messages are destroyed, and zygotic expression takes over to mediate embryonic pattern formation and subsequent development. Mechanistic understanding of this early posttranscriptional regulation of maternally provided transcripts is still fragmentary, partly due to the difficulties in studying RNA-protein interactions and their lack of sequence specificity (Iovino, 2009).

Genomically encoded microRNAs (miRNAs) represent a new layer of posttranscriptional gene regulation that might play an important role in this context. miRNAs bind to specific sequences within the 3'UTRs of mRNAs, leading to degradation of the targeted mRNA or inhibition of protein synthesis. The nature and extent of their role in biological processes are still being debated, but both studies in which miRNA function is abolished wholesale by disrupting their biogenesis and analyses of individual miRNA genes reveal a strong requirement in the control of stem cell fate and in early embryonic development, with higher fishes providing an apparent exception (Iovino, 2009).

In Drosophila, the role of miRNAs in regulating stem cell behavior in the ovaries has been investigated by mosaic analysis of mutants that abrogate miRNA biogenesis. Presumably due to the perdurance of mature miRNAs, mutant clones show age-dependent phenotypes: after 12 days, the number of developing egg chambers is significantly depleted due to reduced division of germline stem cells (Hatfield, 2005); longer-term studies show a gradual loss of both germline and somatic stem cells; in both cases, the underlying causes are unclear (Jin, 2007). Forty-three miRNAs are expressed in the Drosophila germline (Neumüller, 2008), but none of their functions have been described (Iovino, 2009).

This study reports the genomic knockout of the highly conserved miRNA mir-184, which is expressed in the female germline and has assumed control over multiple steps in oogenesis and early embryogenesis in Drosophila. A range of phenotypes of varying penetrance was observed, several of the responsible targets were identified, and their protein levels are were shown to be tuned by miR-184 in vivo. These results support the notion that an individual miRNA can exert phenotypically relevant control over multiple biological processes, and provide insight into the molecular mechanisms of miRNA-mediated regulation in female germline development (Iovino, 2009).

miR-184 was originally identified by expression cloning from the small RNA fraction of Drosophila embryos, but is conserved from insects to humans (Aravin, 2003). Northern analysis shows expression of miR-184 throughout the life cycle, with a relatively weak maternal contribution but strong subsequent zygotic expression; notably, strong expression was found in ovaries. RNA in situ hybridization using the primary transcript as probe shows strong expression in a highly dynamic pattern throughout embryogenesis. miR-184 is also one of the few miRNAs that are expressed in Schneider (S2) cells in significant copy number (Iovino, 2009).

mir-184 is a single copy gene and lies isolated within a 50 kb region on the right arm of the second chromosome. The genomic region is rich in extant P element insertions, including several FRT site-containing elements (PBac{WH}, P-element{XP}; Exelixis Collection), which were used to generate an FLP-induced deletion of 22 kb between the elements PBac{WH}f05119 and P{XP}d08710. To be able to carry out rescue and misexpression experiments, a UAS-mir-184 strain was generated, that contains 1.5 kb of genomic sequence surrounding the mir-184 gene (1 kb upstream, 0.5 kb downstream) (Iovino, 2009).

Δmir-184 zygotic mutant flies eclose at a normal Mendelian ratio and appear morphologically normal, indicating that loss of zygotic expression has no detectable effect on adult viability and no obvious effect on development and overall morphology, which is surprising given the strong and complex expression of the mir-184 transcript throughout embryogenesis. Among adults homozygous for Δmir-184, male fertility is normal; however, females lay far fewer eggs than in wild-type, and the eggs and embryos that are produced show severe abnormalities. Strikingly, the defects become progressively worse over time: young (2- to 3-day-old) Δmir-184 females lay 5-10 eggs per day, which represents <10% of wild-type production. Approximately 70% of the eggs have normal (external) morphology and are fertilized; however, most of these embryos (85%) show severe defects in anteroposterior patterning, and many also show severe defects during cellularization; only about 1% of all progeny develop to adulthood. As the females age, egg production declines further and the number of eggs with an abnormal external morphology increases. Eggs from 3- to 4-day-old females are typically smaller than wild-type, and many show defects in dorsoventral patterning of the egg shell, as judged by the position and length of the dorsal appendages. Δmir-184 females that are 5 days or older lay almost no eggs. Thus, progressive failure of egg production is the prevalent phenotype in the Δmir-184 mutant and supersedes all others within a week. However, its incomplete or delayed penetrance makes it possible to observe a range of distinct other defects as well, indicating that miR-184 function is required for multiple successive steps of oogenesis and early embryogenesis (Iovino, 2009).

The observed phenotypes point to a requirement for miR-184 in either the female germline itself or in the somatic cells of the ovary. RNA in situ hybridization in ovaries is often difficult, and it was not possible to obtain consistent interpretable results when attempting to detect the miR-184 primary transcript. To determine where the requirement lies, UAS-mir-184 transgene was expressed in different cell populations of the ovary using established Gal4 drivers, and under which conditions the sterility phenotype can be rescued was examined, mindful of the possibility that ectopic or even overexpression might lead to phenotypic defects by itself. nos-Gal4VP16 drives expression in the germline cells, C587-Gal4 in most somatic cells of the ovary excluding the cap cells, and GR1-Gal4 drives expression in the follicle cells that envelope the oocyte and produce the egg shell. Expression of mir-184 in the germline (nos-Gal4VP16) strongly rescues the sterility of Δmir-184 females: egg production approaches wild-type levels, and almost all eggs and embryos appear morphologically normal. This indicates that miR-184 is required in the germline, which is consistent with the fact that expression of mature miR-184 is detected in northerns of freshly laid eggs/embryos, that is, prior to the onset of zygotic transcription. Notably, substantial rescue of egg production, although not egg morphology, was observed by simply introducing UAS-mir-184 into the Δmir-184 background. This suggests that, due to the inclusion of 1 kb upstream sequence, the UAS-mir-184 transgene on its own drives moderate expression in the germline. Northern analysis of ovaries from Δmir-184 females that carry the UAS-mir-184 transgene indeed reveals weak expression of mature miR-184, at about 10% of the level observed in wild-type, indicating that the 1 kb upstream sequence included in the UAS construct contains at least part of a germline promoter. Expression of mir-184 in the somatic cell populations of the ovary leads to different results: driving expression using C587-Gal4 has no effect beyond that of UAS-mir-184 alone, whereas driving expression in the follicle cells (GR1-Gal4) leads to severe sterility, suggesting that ectopic or overexpression of mir-184 in follicle cells is in itself detrimental to oogenesis (Iovino, 2009).

miR-184, strongly expressed in the germline and deposited in the egg, regulates several distinct steps during oogenesis and early embryogenesis, including stem cell differentiation and axis formation of both egg chamber and embryo. The underlying molecular mechanism were characterized by identifying three relevant miR-184 targets. Female germline development has long been known to be a carefully regulated process in which the spatiotemporal pattern and activity level of key factors is kept in check by multiple levels of control. The current results show that miR-184 provides a crucial additional layer of regulation. Interestingly, miR-184 does not target the key developmental regulators and morphogens themselves but components involved in their regulation, namely a signal transduction receptor, a transport factor, and a general transcription factor (Iovino, 2009).

Developmentally, the first process miR-184 regulates is the interaction between somatic niche and germline stem cells. Previous genetic analysis of this process has focused on the role of TKV in mediating the DPP signal in stem cell maintenance and cystoblast differentiation. It has now been demonstrated that miR-184-mediated translational repression of SAX protein levels, potentially combined with indirect effects on TKV protein distribution, are a crucial mechanism in dampening DPP signal reception and thus promoting cystoblast differentiation. The substantial rescue of egg production that was observed when halving the gene dose of sax suggests that the lack of cystoblast differentiation (and the subsequent loss of germline stem cells) is responsible for the reduction and ultimate loss of fertility in Δmir-184 mutants (Iovino, 2009).

miR-184's role in establishing egg chamber polarity is more complex. miRNAs have frequently been viewed as performing a clean-up task - suppressing translation of residual transcript after developmental decisions have been made (Giraldez, 2006; Bushati, 2008). The misregulation of K10 in Δmir-184 mutants argues that precocious translation, even within the proper cell (oocyte), may also be deleterious. However, the mechanistic connection between the early overproduction and the later depletion of K10 protein is currently not understood. Because actively translated transcripts are generally considered to be more protected against degradation, a partial loss of K10 transcript seems unlikely. Given that K10 mRNA is bound by translational regulators (Bicaudal D and Egalitarian) and K10 protein interacts with other proteins (Squid), it is possible that these factors themselves are limiting and titrated away by the precocious translation and strong accumulation of K10 protein, but the possibility that other miR-184 targets not yet implicated in dorsoventral patterning of the egg are also involved cannot be excluded (Iovino, 2009).

Finally, in early embryonic development, miR-184 tunes the potent transcriptional repressor TTK69, thereby ensuring the proper timing of pair rule gene expression and anterior-posterior patterning. Several additional phenotypes are readily visible in the mutant that indicate miR-184's involvement in processes known to be tightly regulated, such as the transition into the vitellogenic state, which is stringently controlled by several hormone systems, but also in processes where this is unexpected, such as cortical nuclear migration in the syncytial blastoderm. Detection of the entire range of distinct phenotypes in the Δmir-184 mutant was only possible due to their partial penetrance; however, eventually the requirement for GSC differentiation becomes absolute and, thus, within a week, the loss of egg production supersedes all other phenotypes (Iovino, 2009).

The phenotypes observed in the Δmir-184 mutant partially overlap with those seen in mutants in which miRNA biogenesis is disrupted. However, these experiments are difficult to compare: biogenesis mutants presumably affect all 43 miRNAs normally expressed in the germline, causing additional phenotypes that are likely to epistatically mask effects visible in Δmir-184; in addition, these studies have to be conducted under mosaic conditions, where perdurance of mature miRNAs may add another layer of complication. The polarity and vitellogenesis defects but not the germarium overgrowth that were found in the Δmir-184 mutant have been reported for dcr-1 germline clones. Conversely, dcr-1 germline clones show cell-autonomous cell-cycle defects that were not observed in this study, and GSC maintenance defects cannot be observed in the Δmir-184 mutant, due to its rapid tumorous growth and subsequent regression phenotype (Iovino, 2009).

This study also sheds light on important mechanistic aspects of miRNA function. Most of the defects in the Δmir-184 mutant can be rescued by germline-specific expression of mir-184, indicating that the miRNA is coexpressed with its targets in the same cell and tunes their expression. Loss of mir-184 function leads to increases in protein level in the 2- to 5-fold range, with the mutant showing increased variability in protein level compared to wild-type, concordant with the observed incomplete penetrance and variability in phenotype. The findings support the idea that miRNAs regulate a large number of different targets in vivo. Depending on the stoichiometry and affinity between miRNA and mRNA as well as the critical level of the cognate protein, some of this regulation, although quantifiable at the expression level, may be phenotypically silent. However, the fact that several distinct and molecularly attributable defects are observed in the Δmir-184 mutant clearly indicates that the loss of proper tuning of protein levels frequently becomes phenotypically visible. This is consistent with the longstanding knowledge that many biological processes are sensitive to changes in the activity level of their key components (Iovino, 2009).

Both genetic and molecular analyses demonstrate the key role of the maternal component of miR-184. miR-184 is strongly expressed in the ovaries and later in a highly dynamic pattern throughout embryogenesis, but a pronounced difference was observed in phenotypic impact: loss of the zygotic component has no discernable effect on adult morphology and viability, yet loss from the female germline results in severe morphologic defects in oogenesis and embryonic development. Notably, much of this germline requirement can be rescued by much lower levels of miR-184 than are expressed in wild-type. Moreover, the maternal contribution of miR-184 persists stably through the first 3 hr of development and is then slowly degraded with a half-life of ~ 3 hr. This long perdurance is common to many maternally provided transcripts and typically results in rescue into larval stages and beyond. Thus, it is quite possible that also in the case of miR-184, the persisting maternal contribution rescues whatever zygotic function the miRNA may have, implying that the high level and complex pattern of its embryonic expression might be (partially) redundant (Iovino, 2009).

The remarkable functionality carried by low concentrations of the miRNA highlights the need for complete removal of the maternal contributions of miRNAs when undertaking functional studies. Surprisingly, this consideration has frequently been neglected in current genetic analyses of Drosophila miRNAs, despite the fact that many of those under investigation have weak (similar to miR-184) or even strong maternal contributions (e.g., miR-6 and miR-286). This disregard of maternal contribution and of functional redundancy between family members may be partially responsible for the unusual situation that for Drosophila miRNAs, primarily postembryonic and more subtle phenotypes have been reported, whereas for most vertebrate miRNAs, severe, even embryonic, phenotypes are observed (Iovino, 2009).

Another intriguing finding of this study is that while miR-184 itself is highly conserved, two of the three miR-184 target sites identified are only partially conserved across the Drosophilids, suggesting that the acquisition of molecular targets and thus of regulatory function is in evolutionary flux (Lu, 2008). The fact that poorly conserved sites and even sites with mismatch in the 5' seed region can confer significant and phenotypically relevant repression, draws into question, from a developmental biologist's perspective, the rationale for filtering computational target site predictions based on evolutionary conservation and of applying overly stringent seed matching rules. The results suggest that considering other features of target candidates, such as site accessibility, can provide an important complement to purely sequence-based approaches (Iovino, 2009).

Localized expression pattern of miR-184 in Drosophila

MicroRNAs 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. This study 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 (Li, 2010).

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, the mature nucleotide sequences in human resembles that in mouse completely; only one nucleotide is different between human and fly. 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. To focus future miR-184 function studies, the spatial and temporal expression patterns of miR-184 were determined in Drosophila development (Li, 2010).

First, the temporal expression of miR-184 during Drosophila development was determined. 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, larvae and adult, miRNA-184 became visible at stages 1-4, 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 (Li, 2010).

Second, the expression of miR-184 was determined 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. An analysis of the mature miR-184 by modified ribonuclease protection assay also gave similar results (Li, 2010).

miR-184 shows strong expression in a highly dynamic pattern throughout embryogenesis and is 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. Discernible expression is detected during gastrulation, covering the area of the differentiating neuroectoderm of stage 7 embryos. At late stage 14, miR-184 transcripts are highly enriched, mainly expressed in the central nervous system (CNS) and brain of the embryo (Li, 2010).

Search PubMed for articles about Drosophila MiR-184

Aravin, A. A., et al. (2003). The small RNA profile during Drosophila melanogaster development. Dev. Cell 5: 337-350. PubMed ID: 12919683

Bushati, N., Stark, A., Brennecke, J. and Cohen, S. M. (2008). Temporal reciprocity of miRNAs and their targets during the maternal-to-zygotic transition in Drosophila. Curr. Biol. 18: 501-506. PubMed ID: 18394895

Giraldez, A. J., et al. (2005). MicroRNAs regulate brain morphogenesis in zebrafish. Science 308: 833-838. PubMed ID: 15774722

Hatfield, S. D., et al. (2005). Stem cell division is regulated by the microRNA pathway. Nature 435: 974-978. PubMed ID: 15944714

Iovino, N., Pane, A. and Gaul, U. (2009). miR-184 has multiple roles in Drosophila female germline development. Dev. Cell 17(1): 123-33. PubMed ID: 19619497

Jin, Z. and Xie, T. (2007). Dcr-1 maintains Drosophila ovarian stem cells. Curr. Biol. 17: 539-544. PubMed ID: 17306537

Li, P., et al. (2010). Localized expression pattern of miR-184 in Drosophila. Mol. Biol. Rep. [Epub ahead of print]. PubMed ID: 20339929

Lu, J., et al. (2008). Y. Fu, S. Kumar, Y. Shen, K. Zeng, A. Xu, R. Carthew and C.I. Wu, Adaptive evolution of newly emerged micro-RNA genes in Drosophila. Mol. Biol. Evol. 25: 929-938. PubMed ID: 18296702

Neumüller, R. A., et al. (2008). Mei-P26 regulates microRNAs and cell growth in the Drosophila ovarian stem cell lineage. Nature 454: 241-245. PubMed ID: 18528333

date revised: 2 August 2010

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