Deep sequencing and expression of microRNAs from early honeybee (Apis mellifera) embryos reveals a role in regulating early embryonic patterning
© Zondag et al.; licensee BioMed Central Ltd. 2012
Received: 7 August 2012
Accepted: 22 October 2012
Published: 2 November 2012
Recent evidence supports the proposal that the observed diversity of animal body plans has been produced through alterations to the complexity of the regulatory genome rather than increases in the protein-coding content of a genome. One significant form of gene regulation is the contribution made by the non-coding content of the genome. Non-coding RNAs play roles in embryonic development of animals and these functions might be expected to evolve rapidly. Using next-generation sequencing and in situ hybridization, we have examined the miRNA content of early honeybee embryos.
Through small RNA sequencing we found that 28% of known miRNAs are expressed in the early embryo. We also identified developmentally expressed microRNAs that are unique to the Apoidea clade. Examination of expression patterns implied these miRNAs have roles in patterning the anterior-posterior and dorso-ventral axes as well as the extraembryonic membranes. Knockdown of Dicer, a key component of miRNA processing, confirmed that miRNAs are likely to have a role in patterning these tissues.
Examination of the expression patterns of novel miRNAs, some unique to the Apis group, indicated that they are likely to play a role in early honeybee development. Known miRNAs that are deeply conserved in animal phyla display differences in expression pattern between honeybee and Drosophila, particularly at early stages of development. This may indicate miRNAs play a rapidly evolving role in regulating developmental pathways, most likely through changes to the way their expression is regulated.
A major component of the transcriptome of animals consists of non-protein coding RNAs [1–3]. Micro-RNAs (miRNAs) are a subset of small non-coding RNAs that are 18-24 nucleotides long and have a key role in regulating gene expression in eukaryotes. They are produced from a primary full-length transcript (pri-miRNA), which is cleaved to form hairpin structures around 70 nucleotides in length. These are called precursor miRNAs (pre-miRNAs) and are exported to the cytoplasm to be processed further to functional mature miRNAs by the ribonuclease Dicer [4, 5]. Once assembled into the RNA-induced silencing complex (RISC), the miRNA acts on its target by binding to complementary sequences present in the 3′ untranslated regions (UTR) of the target mRNA . This results in either translational repression or mRNA cleavage, thus providing another level of gene regulation .
There is accruing evidence to suggest that miRNAs play a role in regulating multiple developmental pathways, including fundamental developmental processes of animal development such as axis formation and organ morphogeneis. Many miRNAs are expressed in developmentally restricted patterns [8, 9], for example, examination of miRNA expression in Drosophila during embryogenesis found that many are expressed in restricted patterns along the anterior-posterior and dorso-ventral axes and in specific tissues. Their expression is developmentally regulated, often by their own promoter and regulatory elements, similar to developmental protein coding factors . Loss of miRNA function often results in defective development and patterning [11–14], indicating an essential role in animal development. MiRNAs are also proposed to provide developmental stability particularly under times of environment stress, acting to buffer developmental pathways .
Many miRNA families are ancient and can be traced to more basal animals. Expression analysis and functional studies indicate that their roles are often conserved . As single miRNAs can bind to many different mRNAs to regulate their expression, they can potentially impact on several regulatory pathways . Thus any changes to the way miRNAs are expressed are likely to impact multiple developmental processes. Many miRNAs have been found to be specific to particular phylogenetic groups, some found only in particular lineages. Over 40 miRNA families arose early in the lineage leading to the vertebrates, and it has been suggested that these contributed to the evolution of vertebrate complexity .
Here we have profiled the miRNAs expressed during early embryogenesis in the honeybee (Apis mellifera) to determine if they are likely to play a significant role in honeybee embryogenesis. The expression and function of miRNAs in insect development has to date only been investigated in Tribolium and Drosophila[8, 18]. Like Drosophila, Apis development begins with a syncytial blastoderm stage prior to cellularisation, where much of the body patterning information is established . Both Apis and Drosophila are considered long germ band insects where segmentation occurs across the whole body . However there are some significant differences, notably in patterning of the extraembryonic membranes. In Drosophila the extraembryonic membranes are patterned as one tissue, the amino-serosa, a process regulated by the transcription factor zen . In honeybee, these membranes are patterned separately, although both tissues still require zen . There are also significant differences in the nature of the regulatory networks required to pattern the anterior-posterior axis [22–24]. The honeybee genome has been sequenced and 168 miRNAs have so far been predicted. The Drosophila genome encodes at least 430 miRNAs (mirBase). We examined the expression of honeybee miRNAs during early development, by deep sequencing and in situ hybridization. This included developmental stages at which the anterior-posterior and dorso-ventral axes have been established, patterning including segmentation is underway, just prior to gastrulation. As the pattern of miRNA expression is often reflective of their function in a developmental process or tissue patterning, we examined the expression of eight miRNAs identified in our study. Additionally, RNAi knockdown of Dicer during early embryogenesis indicated that small RNAs are likely to contribute to early honeybee embryo development.
Results and discussion
Abundance and expression of previously known miRNAs
Summary of small RNA sequencing results
Total number of reads
Number of reads mapped back to genome
Number of reads map to annotated pre-miRNA
Profile of known miRNAs in present in 24-30 hour embryos
We determined if these miRNAs are also present in the genomes of other phyla. Of the miRNAs we isolated from honeybee embryos, 36 are also present in the genomes of other arthropods. 15 of these are present in the vertebrate lineage, indicating these are likely to be ancestral (Figure 1B). Less conservation was observed between Apis and Drosophila miRNA content which has also been noted previously between Tribolium and Drosophila, indicating that Arthropod groups (other than Diptera) have more miRNA content in common to each other than when compared separately to Diptera.
To determine if the developmental expression patterns of miRNAs with orthologues in both honeybee and Drosophila were conserved, we examined the expression of four miRNAs detected in our study, with expression and functional data of their orthologue in Drosophila.
In situ hybridisation was performed using RNA probes designed to bind to the pri-miRNA, the longer transcript from which the pre-miRNA is produced, prior to export from the nucleus. This strategy has been successfully used in previous studies illustrating that it reflects the mature miRNA expression (when detected using LNA probes) . The probes used detect nascent transcripts before processing by Drosha RNase III enzyme and thus are expected to detect nuclear dots rather than the cytoplasmic staining produced with probes against an mRNA transcript (for example see Additional file 1: Figure S3).
Previous deep sequencing of Drosophila embryonic RNA revealed that most of the reads correspond to the 3′ arm of the mir-10 precursor , although RNA expression patterns of both mir-10-5p and mir-10-3p is similar in Drosophila embryos . We found that the 90% of sequence reads are from the 5′ arm of the pre-miRNA in honeybee embryos, indicating that mir-10-5p is responsible for the majority of mir-10 function (Figure 1). This has also been found for mir-10 in Tribolium. Changes to which part of the pre-miRNA strand provides the dominant or functional miRNA sequence (arm switching) is proposed to be one mechanism of miRNA evolution to drive miRNA diversification . Our results and those from Tribolium would indicate that the ancestral dominant arm was the mir-10-5p (producing the mature miRNA) and that this has switched during Drosophila evolution to the mir-10-3p arm. However, while only 10% of the total reads for pre-mir-10 were from the 3p arm in honeybee embryos (Figure 1), it was still a significant number (425) and more abundant that some of the other miRNAs detected (Table 2), indicating that the mature miRNA from this arm of the mir-10 hairpin (mir-10-3p) may have a distinct role during honeybee development.
Mir-1 is a highly conserved miRNA that has been suggested to play a role in myogenesis in Drosophila and vertebrates [29–31]. We detected low numbers of reads for Ame mir 1 in both our samples (Table 1), but because of its conservation between vertebrates and invertebrates, we examined its RNA distribution. Staining for pri Ame mir 1 was weakly detected through ventral and anterior mesoderm of the embryo (Figure 2E). By stage 9, expression was found in the anterior of the embryo within the area of the labrum and in restricted regions of the procephalic lobes (Figure 2F). No expression was detected in mesoderm-derived tissues late in development. In Drosophila, Dme mir 1 is also expressed in ventral mesoderm [29, 32] but continues to be expressed throughout mesodermal tissues later in development and is required for muscle and cardiac patterning [29, 33]. This expression pattern of Dme mir 1 is regulated by Twist (Twi), a pro-mesoderm transcription factor . Examination of the upstream and downstream regions surrounding the Ame mir 1 coding region failed to find any significant cluster of Twi binding sites (Additional file 3: Figure S5), possibly explaining the lack of Ame mir 1 expression in the Apis mesoderm. This implies loss of an enhancer element(s) for directed expression in ventral mesoderm in the honeybee, as mesodermal expression is also found in vertebrates suggesting it is a more ancient pattern for mir-1.
Mir-9a is a conserved microRNA in sequence but with differing functions in invertebrates and vertebrates. Drosophila Dme mir 9a is expressed in the dorsal ectoderm and neuro-ectoderm at early stages  and ectodermal epithelial cells including the epithelial surfaces of the head appendages. Dme mir 9a homozygous mutants survive to hatching and are fertile but produce ectopic sensory neurons, indicating a role in negatively regulating neuron number . In vertebrates, however, while mir-9a is expressed in the developing brain it has a differing role, positively regulating neurogenesis [35, 36]. Sequencing data indicated that Ame-mir-9a was expressed (Table 1) during honeybee early development. In situ hybridisation at stage 5, just prior to gastrulation, detected mir-9a throughout the head ectoderm, and then in broad ectodermal stripes across the middle of the embryo to the posterior terminus (Figure 2G). Expression was absent in the dorsal and ventral sides of the embryo (Figure 2G). By stage 9, pri Ame mir 9a RNA was found throughout the epidermis but was weak or absent in neurons of the central nervous system (CNS) (Figure 2H). Expression was strongest in the epidermis of the procephalic lobes and labrum, and no staining was found within cephalic and labrum regions where the neuronal cells are present (Figure 2I). A similar pattern of expression was also found in the mandibles and maxillae, with all appendages exhibiting strong staining around epithelium for pri Ame mir 9a but absent from the central regions of the appendages, where the sensory neuronal tissue are predicted to be (Figure 2J). Therefore mir 9a RNA expression in both honeybee and Drosophila show similar patterns, with strong epithelial cell basis, consistent with a conserved role in regulating production of sensory organ neuronal cells and suppressing sensory neural fate in the surrounding epithelia. In vertebrates, mir-9a has a quite different role in positively regulating neurogenesis, indicating that both its expression and function has changed significantly in the vertebrate group or this developmental role of mir-9a is particular to the insect group.
Mir-184 is also conserved between invertebrates and vertebrates, and has been shown to play an important role in axis formation and oogenesis in Drosophila. Ame-mir-184 had the highest read count of any miRNA in our small RNA library (Table 1). Pri Ame mir 184 RNA was detected in the mesodermal cells throughout the embryo except the dorsal side of the embryo where extraembryonic membranes differentiate (Figure 2K). Previous studies have shown that Dme mir 184 is expressed along the mesoderm on the ventral side of the embryo [32, 37].
Prediction and expression of novel miRNAs
Novel miRNA profile and presence (+) or absence (-) in the genomes of other insects
Knockdown of Dicer during early embryogenesis
We examined the miRNA content of early honeybee embryos by deep sequencing followed by determination of the expression patterns of eight of these miRNAs. Consistent with both miRNA expression patterns and target prediction, Dicer siRNA knockdown embryos had defects in extraembryonic membrane formation, anterior-posterior and dorso-ventral patterning, suggesting that miRNAs may have functions in regulating these patterning pathways or their gene target(s) have roles in multiple pathways.
Many miRNAs that are expressed during embryo development are deeply conserved throughout metazoans indicating a more ancient origin. However, several are unique to the arthropod group, and some restricted just to the Apis lineage. Given the developmental expression of these miRNAs, they may have taken on roles particular to the development of the honeybee embryo. Previous studies have hypothesised that miRNAs are continuously added to the metazoan genomes, are stabilized once added, and are rarely lost [27, 42, 43].
Interestingly, some of the highly conserved miRNAs identified in our study had very different expression patterns in the honeybee to those documented in other animals. Mir 1 expression differed between Apis and other animals, which may result from loss of a regulatory element or binding sites for cis-regulatory proteins . Early embryonic expression of mir 10 differed between Drosophila and Apis, but later expression was similar and is consistent with regulation by separate cis-regulatory elements (one controlling early expression, one controlling later expression) as suggested previously for Drosophila mir-10 . This suggests that earlier expression regulatory elements are evolving more rapidly. A similar pattern of more labile expression in early development has previously observed for protein-coding genes [24, 45–47]. These changes or shifts in miRNA expression imply the regulatory regions controlling miRNA expression are also rapidly evolving. This indicates the importance that changes to gene-regulatory sequences contribute to the evolution of developmental pathways extends to also changes associated with regulatory elements that control miRNA genes.
Sample collection and preparation
A queen honeybee was caged with an empty area of an Eziqueen queen rearing frame and placed back into the hive. After 5 hours the frame was removed and the queen released back into the hive. The eggs were removed and while still attached to the black strips of the Eziqueen frame incubated for 24 hours. Eggs were collected and total RNA extracted using TRIzol (Life Technologies). Total RNA concentration and purity determined was using a Nanodrop spectrometer (Thermo Scientific). 10 μg of purified total RNA was sent to Beijing Genomics Institute (BGI) for sequencing on an Illumina HiSeq 2000 sequencer. Low quality reads, reads without the adaptors, reads with polyA sequences and reads without the insert tag where removed. Also discarded were any tRNA, rRNA, snRNA and snoRNA sequences. Sequence reads were mapped to the genome using the programme SOAP . Small RNA tags were aligned to known miRNA Apis mellifera precursors (miRBASE). The two small RNA libraries shared 98.06% of sequences. Analyses of the length distribution of cleaned reads showed enrichment of small RNAs from 22 to 31 nucleotides (Additional file 7: Figure S1); we would expect about a length of 22nt for miRNAs. Examination of the first base of the 22 nucleotide sequences revealed most show a first base bias to uridine as predicted from previous deep sequencing miRNA studies (Additional file 8: Figure S2).
Amplication of pri-miRNA fragments
Oligonucleotide primers were designed to amplify 500-800 bp regions using genomic template. In each amplicon, the precursor miRNA resides in the centre of the sequence. PCR fragments were cloned into the vector pBluescript II KS (+/-) and sequenced. Cloned fragments were used to produce RNA sense and antisense in situ hybridisation probes. Oligonucleotide primer pairs were as follows: mir-10 5′ACAAATGGACGACGAAGAGG3′ and 5′GCGGCACGTACGTTACTTTA3′, mir-1 5′GCCACGTACGTTCGAAAACT3′ and 5′TTCGCAAGACGGATACATCA3′, mir-184 5′ GCCTCGGGTTTCGAGGCGTT3′ and 5′ AGGAGAAGGGAAGAATGTGCAGAGA3′, mir-9a 5′CCGATTTCTCCGTCTTTTCTG3′ and 5′CCGATTTCTCCGTCTTTTCTG3′, mir-0002 TGTACGGGCAGTACTGGG and TCTTGATGATGCGTCTTG, mir-0004 5′CAACGATGCGTTTCGACTTA3′ and 5′GTACCCACGAGTCGTCAC3′, mir-0005 5′TCGATATTCGAAACGCAACA3′ and 5′TGGATTTGAATTCGTGTATGAAA3′, mir-0007 5′ACGAGGATACACGGATGGAC3′ and CAATTCACTTCCTTTTCACCTCA3′.
In situhybridization on honeybee embryos
Performed as per Osborne  with the following modifications. Incubations with pri-miRNA anti-sense and sense probes were carried out at 60°C with rotation for 48 hours before post-hybridisation wash steps to remove excess probe. Embryos were incubated overnight at 4°C with anti-dioxygenin-alkaline phosophatase antibody with rotation before post-antibody wash steps and colour reaction.
Dicer siRNA knockdown
Two siRNAs were designed against Am-dicer; GGACGAAGAGUUAGAGUUAUU and UGAAACAGCUAGUGAUAUAUU. The two siRNAs were injected together at a final concentration of 5 μg/ml into freshly laid eggs attached to plastic strips from an Eziqueen frame . As a control, a non-targeting siRNA (D-001810-01-05, Dharmacon) was also injected at the same concentration. Following injection, embryos were placed in a humidified incubator at 35°C for 48 hours. After incubation they were fixed with heptane/formaldehyde in PBS overnight, rocking at room temperature. After fixation, embryos were washed with PBS and stained with DAPI before visualization on an Olympus BX61 microscope with a DP71 camera. Embryos were staged as per DuPraw .
We would like to thank James Smith for critical reading of this manuscript. Otto Hyink for help with the honeybee hives. Cris Anderson for technical assistance. This work was supported by a University of Otago Research Grant to MJW.
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