The iab-4 noncoding RNA from the Drosophila bithorax complex is the substrate for a microRNA (miRNA). Gene conversion was used to delete the hairpin precursor of this miRNA; flies homozygous for this deletion are sterile. Surprisingly, this mutation complements with rearrangement breakpoint mutations that disrupt the iab-4 RNA but fails to complement with breaks mapping in the iab-5 through iab-7 regulatory regions. These breaks disrupt the iab-8 RNA, transcribed from the opposite strand. This iab-8 RNA also encodes a miRNA, detected on Northern blots, derived from the hairpin complementary to the iab-4 precursor hairpin. Ultrabithorax is a target of both miRNAs, although its repression is subtle in both cases (Bender, 2008).
A large number of microRNA (miRNA) clones prepared from Drosophila RNA have been characterized at a variety of developmental stages. Two of these clones matched sequences from the BX-C, mapping to the 3' end of a ncRNA discovered by Cumberledge (1990). This ncRNA was called the 'iab-4 RNA,' because it was thought to come from the iab-4 segmental domain of the BX-C, and the miRNAs were named miR-iab-4 5p (five prime) and miR-iab-4 3p (three prime). More recent mapping of segmental domains (Bender, 2000) has shown that the RNA actually lies in the iab-3 domain (regulating parasegment 8), and indeed, the ncRNA is expressed from parasegment 8 through parasegment 12 (Cumberledge, 1990). However, the iab-4 nomenclature is maintained here to avoid confusion with the designations in other studies. Two cDNA clones for the iab-4 RNA were described by Cumberledge (1990), with alternate 3' poly(A) sites separated by 304 base pairs (bp). The two miRNAs come from this region between these two poly(A) sites; both are presumably derived from a 70-base hairpin RNA precursor predicted from the sequence (Bender, 2008).
A recent study (Ronshaugen, 2005) suggested that the miR-iab-4 5p miRNA might be responsible for repression of Ubx in the abdominal segments where the miRNA is expressed. The conclusion was based on experiments in which miR-iab-4 5p was expressed at high levels in tissues, including the wing and haltere discs, where miR-iab-4 5p is not normally found. However, the pattern of UBX expression in PS8, where the miRNA is expressed, is very similar to the UBX pattern in PS7, which lacks the miRNA. The obvious repression of Ubx in both of these parasegments is clearly dependent on abd-A; any effect of miR-iab-4 5p must be subtle or redundant. Moreover, misexpression of miRNAs in other systems have been shown to give misleading effects. The function of miR-iab-4 5p can best be examined by mutating or deleting the miRNA (Bender, 2008).
Prior studies characterized a large number of P element insertions in the BX-C, including one called HCJ200, which maps only ~200 bp proximal to the miRNAs. This provided the opportunity to mutate the miRNAs by P-element-mediated gene conversion. Loss of the miRNAs derived from the iab-4 ncRNA causes no apparent morphological or behavioral phenotype, but the analysis revealed a functional miRNA derived from the opposite strand (Bender, 2008).
A 3.7-kb conversion donor fragment was constructed with a mutated version of the miRNA precursor sequence. The precursor sequence is symmetrically cut by the BstZ17I restriction endonuclease; this permitted the replacement of most of the precursor sequence with a double-stranded oligonucleotide-containing sites for the HindIII and I-SceI endonucleases. A plasmid with the donor sequence and a plasmid to supply P-element transposase were both injected into embryos with the HCJ200 (rosy+) P insertion. Offspring from the injected individuals were screened for loss of the HCJ200 P element (i.e., rosy-), and progeny from these exceptional flies were screened by PCR for a change in the size of the genomic DNA at the site of the insertion. One of 86 fertile injected animals gave the expected convertants. The conversion events were verified by sequencing the PCR product, and by whole-genome Southern blots. Genomic DNA was cut with HindIII, which cuts the oligonucleotide introduced in the conversion but does not cut the wild-type sequence within the 3.7-kb donor fragment. The sizes of the HindIII fragments on the Southern blot confirmed that the donor sequence was at the expected position in the BX-C and not at another genomic location (Bender, 2008).
Flies homozygous for the conversion chromosome (henceforth called 'δ') appeared normal. In particular, no evidence of segmental transformation was seen in mounted adult abdominal cuticles of either sex. However, both sexes were sterile. Females had ovaries with eggs of normal size, but only very rare individuals ever laid an egg, even after mating with wild-type males, and these rare eggs never hatched. Males had morphologically normal testes containing motile sperm. In single-fly tests for mating behavior, δ homozygous females mated with wild-type males as readily as their heterozygous siblings. The δ homozygous males showed normal courtship behavior toward wild-type females, except that they never completed copulation. The mutant males mounted the females, but they did not bend their abdomens quite far enough to mate. Thus, the sterility in both sexes appeared to be behavioral, due to a defect in the nerves or muscles required to lay eggs or to curl the abdomen (Bender, 2008).
The δ mutation was tested for complementation with rearrangement mutations in the BX-C, including several that should disrupt the iab-4 RNA transcript upstream of the position of the miRNA precursor. Surprisingly, breaks disrupting the iab-4 transcription unit complemented with δ -- i.e., trans-heterozygotes were fertile. Thus, the iab-4 RNA is not the precursor for any miRNA that is responsible for fertility. In contrast, rearrangements distal to the position of the miRNAs failed to complement, even with breaks >50 kb distal. Assuming that noncomplementing rearrangements are upstream in the precursor, one can deduce that the precursor is transcribed distal to proximal on the chromosome, and that the miRNA responsible for fertility comes from the opposite strand to those detected by Aravin (2003). Similarly, one would predict that the precursor transcript for the fertility miRNA spans at least the iab-7 through the iab-3 segmental domains (Bender, 2008).
Several studies have detected such a transcript (Sánchez-Herrero, 1989; Zhou, 1999; Bae, 2002; Bender, 2002), which is usually called the iab-8 ncRNA. It has been detected and mapped solely by in situ hybridization to RNA in embryos, although the complementation analysis now corroborates the in situ mapping, at least for the iab-4 through iab-7 region. Its start site is near Abd-B; it was detected by probes 4 kb proximal to the 3' end of the Abd-B transcripts. Zhou (1999) defined a potential promoter for the iab-8 transcript, which lies ~5 kb downstream from Abd-B, although the evidence did not preclude a start site further upstream. Bae (2002) detected the iab-8 RNA upstream of the Abd-B class A RNA start site (the 'BPP' probe, suggesting a more distal start site. However, hybridization to the Abd-B class B RNA in the ninth abdominal segment (PS14) could have been mistaken for the iab-8 RNA pattern. Moreover, the iab-8S10 breakpoint, just proximal to Abd-B, does complement the sterility phenotype of δ, and so the promoter for the iab-8 fertility function should be to the left of that break (Bender, 2008).
At the 3' end, the iab-8 RNA extends through abd-A. The iab-8 RNA in situ pattern was detected by a probe 5.5 kb proximal to the 3' end of the abd-A poly(A) site. Thus the transcription unit spans at least 120 kb. The iab-8 RNA has not been detected by probes in the bxd regulatory region, further proximal to abd-A (Bender, 2008).
The iab-8 transcript initiates at the cellular blastoderm stage (Zhou, 1999), as do most of the other embryonic ncRNAs (Akam, 1985; Cumberledge, 1990; Bae, 2002). However, it should take ~45 min to transcribe to the position of the miRNA precursor hairpin, assuming a transcription speed of ~1.3 kb/min. This would account for the developmental delay in the appearance of the RNA signal. The iab-8 RNA is located in the eighth abdominal segment and in more posterior segmental rudiments. In late embryos, the transcript persists in the posterior end of the ventral nerve chord (Bender, 2008).
Embryos homozygous for the δ mutation showed no apparent changes in the patterns of ABD-A and ABD-B, but there were subtle differences in the UBX pattern. UBX is expressed strongly and comprehensively in the cells of parasegment 6 (PS6, primarily the first abdominal segment). In the second abdominal segment (PS7), ABD-A appears and turns off UBX, especially in the more anterior cells of the parasegment. In the more posterior segments, the UBX staining pattern weakens progressively, and the ABD-A pattern becomes somewhat stronger. However, in δ embryos, the UBX pattern is nearly constant from PS7 through PS12. Thus, the progressive posterior decline in wild-type embryos appears not to be caused by ABD-A or ABD-B but rather by miR-iab-4 5p, whose expression shows a progressive posterior increase in PS8-12 (Bender, 2008).
In the eighth abdominal segment (PS13) of a wild-type embryo, UBX is almost completely absent in both the epidermis and the CNS. Homozygotes for δ show a partial derepression of UBX in the CNS in PS13. The derepression is similar in pattern and intensity to that seen in AbdB-/+ heterozygotes (data not shown). The repression of Ubx could be indirect; miR-iab-8 could be a positive regulator of Abd-B (and miR-iab-4 a positive regulator of abd-A). But all known targets of miRNAs are negatively regulated, and so it seems more likely that both miRNAs directly regulate Ubx (Bender, 2008).
It is possible that the effects of these two miRNAs are masked by functional redundancy with abd-A (for miR-iab-4) and Abd-B (for miR-iab-8). Embryos lacking ABD-A (but retaining miR-iab-4) show a dramatic derepression of UBX in the second through seventh abdominal segments. There does appear to be a slight decline in UBX levels in the more posterior segments, which could be attributed to miR-iab-4 repression. A complete analysis would include the UBX expression in embryos lacking both abd-A and miR-iab-4, but that will require construction of an abd-A, δ double mutant chromosome. In any case, miR-iab-4 repression of Ubx is subtle, even in the absence of ABD-A. Similarly, in an Abd-B homozygous mutant embryo (retaining miR-iab-8), UBX expression in the eighth abdominal segment closely resembles that in the seventh. Again, a δ, Abd-B double mutant chromosome would be useful for comparison, but again it is clear that the repression of Ubx by miR-iab-8 is still subtle in the absence of ABD-B. There is no reason to expect that Ubx is the only target of these miRNAs; perhaps other target genes will be discovered which the miRNAs repress more dramatically (Bender, 2008).
MiR-iab-8 is the first example of a functional product of a ncRNA in the BX-C. There are no other predicted miRNA precursor sequences in the iab-8 RNA or elsewhere in the BX-C (the Antennapedia complex includes miR-10), but there are many other ncRNAs. The possibility that they also include functional products now seems more likely (Bender, 2008).
Many microRNA loci exhibit compelling hairpin structures on both sense and antisense strands; however, the possibility that a miRNA gene might produce functional species from its antisense strand has not been examined. Antisense transcription of the Hox miRNA locus mir-iab-4 generates the novel pre-miRNA hairpin mir-iab-8, which is then processed into endogenous mature miRNAs. Sense and antisense iab-4/iab-8 miRNAs are functionally distinguished by their distinct domains of expression and targeting capabilities. miR-iab-8-5p, like miR-iab-4-5p, is also relevant to Hox gene regulation. Ectopic mir-iab-8 can strongly repress the Hox genes Ultrabithorax and abdominal-A via extensive arrays of conserved target sites, and can induce a dramatic homeotic transformation of halteres into wings. The antisense miRNA principle is generalizable: it has been shown that several other loci in both invertebrates and vertebrates are endogenously processed on their antisense strands into mature miRNAs with distinct seeds. These findings demonstrate that antisense transcription and processing contributes to the functional diversification of miRNA genes (Tyler, 2008).
These studies of BX-C miRNAs reveal two principal insights into miRNA regulatory biology. First, a new Hox cluster miRNA, mir-iab-8, was identified. Using gain-of-function assays it was shown that it can strongly inhibit Ubx and abd-A and generate homeotic phenotypic transformations. Indeed, the Hox-regulatory properties of mir-iab-8 are far more potent than those of mir-iab-4 (Ronshaugen, 2005), and correlate directly with the properties of its target sites in their 3' UTRs. Curiously, both BX-C miRNAs obey organizational and functional rules previously defined for the protein-encoding members of the BX-C. These regulatory RNAs exhibit colinearity, in that transcription of pri-mir-iab-8 initiates more distally on the chromosome and is expressed more posteriorly in the embryo relative to pri-mir-iab-4. They also exhibit posterior prevalence, in that both sense and antisense iab-4 miRNAs directly repress multiple homeotic genes located more anteriorly in the Hox cluster. In fact, the next most-anterior Hox gene Antp is a third likely endogenous iab-miRNA target that contains highly conserved target sites with t1A features for miR-iab-8-5p. In contrast, Abd-B contains no conserved sites for either iab-4 or iab-8 miRNAs in its long (>2 kb) 3' UTR. Therefore, BX-C miRNAs and homeobox genes are governed by the same regulatory logic (Tyler, 2008).
It is worth recalling that saturation mutagenesis screens of the BX-C revealed only three loci that are required for viability and exhibit homeotic defects, corresponding to the homeobox genes Ubx, abd-A, and Abd-B. In contrast, pioneering studies by Lewis (1978) considered rearrangements and dominant alleles suggested the existence of at least eight homeotic 'factors' in this region of the genome. Although many of these are now recognized as cis-regulatory elements that regulate Hox gene transcription, this work with BX-C miRNAs reveals two bona fide Hox regulators that are capable of inducing severe dominant homeotic transformations. The endogenous requirement for iab-4/iab-8 miRNAs appears to be subtle, possibly due to compensatory transcriptionally based regulatory mechanisms. Nevertheless, loss of function analysis corroborates that these miRNAs are required for normal expression of Hox targets in the nervous system and for normal development. These data emphasize that loss-of-function and gain-of-function genetics are complementary approaches to uncover important regulatory molecules (Tyler, 2008).
Antisense transcription and processing were uncovered in this study as a mechanism to generate new functional miRNAs. Bioinformatic analysis suggests that a large fraction of miRNA loci are theoretically competent to produce antisense miRNAs. Extant cloning efforts suggest that few miRNA loci actively produce large quantities of antisense miRNAs. Nevertheless, the sequencing effort reported in this study has revealed additional instances of putative antisense miRNAs. Although none of these was cloned more than twice, genetics demonstrates that rare miRNAs (e.g., lsy-6, expressed by a handful of neurons in a whole animal) can be critical components of regulatory networks and can have potent biological activities. Therefore, assessment of the biological relevance of the other antisense miRNA candidates awaits further study (Tyler, 2008).
In the case of the iab-4 locus, the regulatory diversity afforded by sense and antisense transcription of a single miRNA hairpin is manifested by altering the seed regions of their respective miRNA products and by deploying the sense and antisense pri-miRNA transcripts in distinct spatial domains. It might in fact be deleterious for a given locus to be simultaneously transcribed on both strands -- either because of transcriptional interference from colliding polymerase complexes, or because of the possibility to inadvertently generate dsRNA. Further analysis is needed to test the notion that it is favorable for sense/antisense miRNA pairs not to be expressed in the same cells. Overall, though, as animal genomes are quite extensively transcribed, and many miRNA genes adopt extensive hairpins on both strands, the potential for endogenous antisense processing of miRNA hairpins is theoretically quite broad. It is proposed that antisense transcription of other miRNA loci might generate novel small RNAs whose potentially beneficial regulatory activities are available for selection and stabilization by natural selection. This identification of several confident examples of antisense miRNAs, whose processing and/or targets have been conserved among diverse species, provides compelling support for this hypothesis (Tyler, 2008).
Computational studies have identified the Ubx 3' UTR as a likely target of regulation by iab-4-5p (Stark, 2003; Grun, 2005). Of the seven potential sites identified by Stark, five exhibit conserved and canonical seed pairing of six or more nucleotides. Of these, sites #3 and #6 are perfectly conserved among sequenced Drosophilids and have seeds of at least 7 nt, a length sufficient for efficient in vivo recognition by miRNAs; site #7 also has a 7-mer seed match that is conserved in some species (Ronshaugen, 2005).
In current target-finding approaches, greater confidence is usually ascribed to those miRNA-binding sites that are conserved in the greatest number of analyzed species. Curiously, the putative iab-4-5p target sites with the lowest free energy are not necessarily the best conserved. Instead, there appear to be compensatory changes among different iab-4-5p-binding sites in individual Ubx 3' UTRs. For example, site #4 exhibits canonical 6-mer seed pairing in four species of Drosophila, but contains a G:U base pair in Drosophila virilis and a seed mismatch in Drosophila mojavenesis and is likely nonfunctional in these two species. Conversely, site #7 is mispaired in D. melanogaster and Drosophila yakuba, but is conserved as a strong 7-mer seed-paired site in D. mojavenesis, Drosophila pseudoobscura, Drosophila ananassae, and D. virilis. These observations suggest that individual target sites may be evolutionarily labile, and in vivo regulation depends on the net complement of both high- and low-affinity sites contained in the target mRNA. These compensatory changes in strong and weak target sites are reminiscent of the evolution of individual Bicoid-binding sites in the eve stripe 2 enhancers present in divergent Drosophilids (Ronshaugen, 2005).
Direct evidence for iab-4:Ubx miRNA interactions was obtained using a tub::GFP-Ubx 3' UTR transgene (the "Ubx sensor"). This construct directs ubiquitous expression of the GFP coding sequence fused to the Ubx 3' UTR, and wing imaginal discs bearing the Ubx sensor display relatively uniform expression of GFP. Ectopic expression of UAS-DsRed under the control of ptc-Gal4, which directs expression along the anterior-posterior border of the disc, has little or no effect on the distribution of GFP staining (Ronshaugen, 2005).
The expression of the Ubx sensor was assayed in the presence of ectopic iab-4 miRNAs. For this purpose, a transgene was created that contains DsRed and 400 base pairs (bp) from iab-4 encompassing the entire 100-bp 3' hairpin sequence (UAS-DsRed-iab-4). Transgenes of this type direct the expression of biologically active miRNAs in cells that are labeled by expression of DsRed (Stark, 2003). When driven by ptc-Gal4 in wing imaginal discs, Ubx sensor levels were specifically diminished in those cells expressing the iab-4 transgene. Detailed analysis of the DsRed-iab-4 and GFP-Ubx expression profiles suggests that repression of the Ubx sensor by ectopic iab-4 miRNA is dose-sensitive. These data constitute in vivo evidence that iab-4 miRNAs specifically recognize target sequences in the Ubx 3' UTR and thereby attenuate Ubx protein synthesis (Ronshaugen, 2005).
Ubx protein is broadly distributed throughout the haltere imaginal disc, where it imposes haltere identity by repressing the expression of many genes that otherwise direct wing development. This repression is very sensitive to Ubx levels, and consequently, even partial loss of Ubx function can transform halteres into wings. Haltere discs were examined for the accumulation of Ubx protein in the absence or presence of ectopic iab-4 miRNAs. Ubx is detected at high levels in most of the cells of the presumptive pouch. Expression of DsRed alone using bx-Gal4, which is active in the presumptive dorsal region of the pouch, did not affect Ubx accumulation. In contrast, haltere discs expressing UAS-DsRed-iab-4 under the control of bx-Gal4 displayed strongly reduced levels of Ubx protein. Thus, as seen for the Ubx sensor in wing discs, ectopic iab-4 miRNA inhibits accumulation of endogenous Ubx protein (Ronshaugen, 2005).
The effect of iab-4 miRNA misexpression on adult haltere development was examined. The wild-type haltere contains small lightly pigmented sensilla but lacks the triple row of sensory bristles at the leading margin seen in wings. In contrast, halteres that developed from discs expressing UAS-DsRed-iab-4 under the control of bx-Gal4 or scalloped-Gal4 are flattened and elongated in the proximal-distal axis, and exhibit an extensive row of sensory bristles at the leading margin. All of these phenotypes are strongly indicative of a classic haltere-to-wing homeotic transformation (Ronshaugen, 2005).
The demonstration that miR-iab-4 represses the anterior Hox gene Ubx might be relevant to the phenomenon of 'posterior prevalence'. Polycomb mutant embryos have previously been observed to derepress Hox gene expression, resulting in broad misexpression of all Hox genes. Ultimately, ectopic expression of posterior Hox genes (e.g., Abd-B or Hox9-13) leads to the transcriptional repression of anterior Hox genes (e.g., Ubx or Hox8 paralogs). Polycomb mutant embryos also derepress iab-4 expression throughout the embryo. Therefore, misexpression of iab-4 miRNAs may contribute to the repression of Ubx function observed in the Polycomb mutant background. Thus, posterior prevalence may arise from the dual utilization of protein-based/transcriptional mechanisms and miRNA-based/post-transcriptional mechanisms (Ronshaugen, 2005).
In Drosophila melanogaster, the iab-4/iab-8 locus encodes bi-directionally transcribed microRNAs that regulate the function of flanking Hox transcription factors. This study showed that bi-directional transcription, temporal and spatial expression patterns and Hox regulatory function of the iab-4/iab-8 locus are conserved between fly and the beetle Tribolium castaneum. Computational predictions suggest iab-4 and iab-8 microRNAs can target common sites, and cell-culture assays confirm that iab-4 and iab-8 function overlaps on Hox target sites in both fly and beetle. However, w key differences were observed in the way Hox genes are targeted. For instance, abd-A transcripts are targeted only by iab-8 in Drosophila, whereas both iab-4 and iab-8 bind to Tribolium abd-A. This evolutionary and functional characterization of a bi-directionally transcribed microRNA establishes the iab-4/iab-8 system as a model for understanding how multiple products from sense and antisense microRNAs target common sites (Hui, 2013).
The iab-4 miRNA locus has some unusual properties: the locus is transcribed in both directions, producing two primary miRNA transcripts and two hairpin precursors. Each precursor is processed to produce two mature miRNAs, one from each arm of each precursor hairpin. Only a handful of other miRNAs have been shown by deep sequencing data to be transcribed in both directions; currently, the miRBase database has only 27 animal examples. This study shows that bi-directional transcription of the iab-4/8 locus and production of miRNAs from both transcripts is conserved in insects. However, the relative abundance of the four mature miRNAs varies significantly between fly and beetle (Hui, 2013).
The four mature miRNAs produced from the iab-4 locus are extremely similar. Indeed, they are all seed-shifted variants of each other. This state is possible only because the mature sequences are partially palindromic. Thus, sense and antisense sequences are highly similar, as are partially complementary mature sequences from opposite arms of the same hairpin. As the mature sequences are closely related, the predicted targets of the four mature products overlap significantly. Previous work suggests that this is an unusual situation: the targets of alternate miRNAs derived from the 5'- and 3'-arms of almost all miRNAs are largely different. It was shown that iab-4-5p and iab-8-5p have more common targets that expected by chance. This functional overlap of antisense products may have facilitated the maintenance of the bi-directionality in the iab-4/iab-8 locus. Indeed, the same pattern was observed in mir-307, the other miRNA locus that produces mature miRNAs from both genomic strands (Hui, 2013).
This analysis of the repression of engineered perfect target sites clearly shows significant cross-targeting for three of the four mature miRNAs. Furthermore, it was shown that the Hox gene Ubx/Utx is a conserved target of both iab-4 and iab-8 miRNAs in both Drosophila and Tribolium. However, between fly and beetle, differences were found in Hox gene targets of iab-4/8 miRNAs and differences in the sites that mediate those targets. For example, abd-A is regulated only by iab-8 miRNAs in Drosophila, whereas both iab-4 and iab-8 miRNAs target abd-A transcripts in Tribolium. There are, therefore, both conserved and variable aspects of the targeting properties of the four mature miRNAs produced from the iab-4/8 locus in insects. The conservation of the Hox genes Ubx and abd-A as targets of the iab-4/8 miRNAs further establishes the ancient connection between the miRNAs of the Hox complex and their role in modulating the function of the Hox genes themselves. No other intergenic miRNA has been linked by genomic position to its function, yet all Hox complex miRNAs (iab-4, mir-196 and mir-10) have been found to modulate Hox gene function (Hui, 2013).
The iab-4/8 locus provides for fundamental insight into the mechanisms of evolution and the function of sense/antisense miRNA pairs. The production of functional products from both strands of the same locus may impose an evolutionary trade-off, driven on one hand by sequence conservation because of structural constraints, and on the other hand by constraints imposed by target specificity. It is proposed that the deep conservation can be explained in part by the common targeting properties of the multiple mature products generated from these two transcripts. Given the functional similarity of the miRNA products of iab-4 and iab-8, the antisense transcription of the locus can be considered analogous to the acquisition of an enhancer by the sense transcript to drive expression and miRNA production in the additional domain. Furthermore, the palindromic nature of the iab-4/iab-8 mature sequences determines that the novel antisense miRNA will share targets with the pre-existing sense miRNA. It is suggestd that this explains the apparent contradiction between extreme conservation of mature miRNA sequences on both arms, yet significant plasticity between organisms as to which arm is the dominant product. However, the evolution of target sites in abd-A demonstrates that functional target sites can be differentially regulated between even closely related species (Hui, 2013).
The relationship between microRNA regulation and the specification of behavior is only beginning to be explored. This study finds that mutation of a single microRNA locus (miR-iab4/8) in Drosophila larvae affects the animal's capacity to correct its orientation if turned upside-down (self-righting). One of the microRNA targets involved in this behavior is the Hox gene Ultrabithorax whose derepression in two metameric neurons leads to self-righting defects. In vivo neural activity analysis reveals that these neurons, the self-righting node (SRN), have different activity patterns in wild type and miRNA mutants while thermogenetic manipulation of SRN activity results in changes in self-righting behavior. These data thus reveal a microRNA-encoded behavior and suggests that other microRNAs might also be involved in behavioral control in Drosophila and other species (Picao-Osorio, 2015).
The regulation of RNA expression and function is emerging as a hub for gene expression control across a variety of cellular and physiological contexts, including neural development and specification. Small RNAs such as microRNAs (miRNAs) have been shown to affect neural differentiation, but their roles in the control of behavior are only beginning to be explored (Picao-Osorio, 2015).
Previous work has focused on the mechanisms and impact of RNA regulation on the expression and neural function of the Drosophila Hox genes. These genes encode a family of evolutionarily conserved transcription factors that control specific programs of neural differentiation along the body axis, offering an opportunity to investigate how RNA regulation relates to the formation of complex tissues such as the nervous system (Picao-Osorio, 2015).
This study used the Hox gene system to investigate the roles played by a single miRNA locus (miR-iab4/iab8) on the specification of the nervous system during early Drosophila development. This miRNA locus controls the embryonic expression of posterior Hox genes. Given that no detectable differences were found in the morphological layout of the main components of the nervous system in late Drosophila embryos of wild type and miR-iab4/iab8-null mutants [herein ΔmiR], this study analyzed early larval behavior as a stratagem to probe the functional integrity of the late embryonic nervous system (Picao-Osorio, 2015).
Most behaviors in early larva were unaffected by the miRNA mutation, except self-righting (SR) behavior: miRNA mutant larvae were unable to return to their normal orientation at the same speed as their wild-type counterparts (Picao-Osorio, 2015).
By means of selective target overexpression followed by SR phenotype analyses, this study identified the Drosophila Hox gene Ultrabithorax (Ubx) as a miRNA target implicated in the genetic control of SR behavior. Overexpression of Ubx within its expression domain did not affect any larval behavior tested except SR, which is in agreement with the effects observed in miRNA mutants. Analysis of Ubx 3' untranslated region (3'UTR) fluorescent reporter constructs expressed in the Drosophila central nervous system (CNS) indicates that the interaction between miR-iab4/iab8 and Ubx is direct, which is in line with prior observations in other cellular contexts (Picao-Osorio, 2015).
To identify the cellular basis for SR control, Ubx was systematically overexpressed within subpopulations of neurons. Increased levels of Ubx within the pattern of Cha(7.4kb)-Gal4, which largely targets cholinergic sensory and interneurons, phenocopied the miRNA SR anomalies. Further overexpression analysis identified two metameric neurons as the minimal node required for the SR behavior [self-righting node (SRN)] (Picao-Osorio, 2015).
Several lines of evidence confirm the role of miRNA-dependent Ubx regulation within the SRN as a determinant of SR. First, both Ubx and miRNA transcripts (miR-iab4) derived from the miR-iab4/iab8 locus were detected within the SRN. Second, in the context of miRNA mutation, Ubx protein expression is increased within the SRN. Third, reduction of Ubx (Ubx RNAi) specifically enforced within SRN cells is able to ameliorate or even rescue the SR phenotype observed in miRNA mutants (Picao-Osorio, 2015).
Two plausible scenarios arise to explain the effects of miR-iab4/iab8 in regard to SR behavior. One is that miRNA input is required for the late embryonic development of the neural networks underlying SR, arguing for a 'developmental' role of the miRNA; another is that miRNA repression affects normal physiological/behavioral functions largely without disrupting neural development in line with a 'behavioral' role. Two independent experiments support that the primary roles of miR-iab4/8 are behavioral. First, anatomical analysis of SRN cells in wild type (wt), ΔmiR, and R54503>Ubx [SRN-driver line] show no significant differences in total numbers of SRN cells or in SRN cell body size; furthermore, analysis of wt, ΔmiR, and R54503>Ubx show indistinguishable SRN-projection patterns. Second, Gal-80ts-mediated conditional expression experiments show that SRN-specific Ubx overexpression after embryogenesis is sufficient to trigger the SR behavior (Picao-Osorio, 2015).
These results suggest that miRNA-dependent Hox regulation within the SRN must somehow modify the normal physiology of SRN cells so that when the miRNA is mutated, these neurons perform functions different from those in wild-type animals. To test this hypothesis, genetically encoded calcium sensors [GCaMP6] specifically expressed in SRN cells were used, and spontaneous profiles of neural activity were tracked down. SRN cells in miRNA mutants produce activity traces that are significantly different from those observed in wild-type SRN cells. Quantification of maximal amplitude and proportion of active cells in each genotype also reveal significant differences in SRN function across the genotypes, but no change in cell viability is observed. Neural activity differences across genotypes are significant within regions of expression of miR-iab4, suggesting that this miRNA (and not miR-iab8) might be the main contributor to SR control. Analysis of mutations that selectively affect miR-iab4 or miR-iab8 strongly suggests that miR-iab4 is the key regulator of SR (Picao-Osorio, 2015).
To demonstrate that the changes in SRN neural activity were causal to SR behavior, SRN cells were artificially activated or inhibited this was shown to trigger the aberrant SR phenotype. This suggested that activation of SRN cells in larvae placed 'right side up' might be sufficient to 'evoke' actions reminiscent of a self-righting response. An optogenetic system was developed in which SRN cells were activated by means of R54F03-driven channelrhodopsin 2 (ChR2) in trans-retinal fed larvae. Under blue light stimulation, larvae performed an atypical bending movement, frequently adopting a 'lunette' position. Neither parental line R54F03-Gal4 nor UAS-Ch2R showed similar reactions to stimulation, confirming the specificity of this effect (Picao-Osorio, 2015).
To study the links between SRN neurons and the SR movement, SRN projections were labeled with myr-GFP and SRN cells were discovered to innervate two of the lateral transverse (LT) muscles and can be colabeled antibodies against Fasciclin 2 (Fas2), demonstrating these to be motorneurons. LT muscles are innervated by Bar-H1+ motorneurons, so Bar-H1-Gal4 was used as a second driver to demonstrate that appropriate Ubx levels in these cells are required for normal SR behavior, establishing the SRN cells as the LT-MNs (Picao-Osorio, 2015).
This study has therefore shown that miRNA-dependent Hox gene repression within a distinct group of motorneurons (SRN/LT-MNs) is required for the control of a specific locomotor behavior in the early Drosophila larva. The finding that Hox gene posttranscriptional regulation is involved in SR control suggests that other RNA-based regulatory processes affecting Hox gene expression might also impinge on specific neural outputs; this possibility is currently being investigated, with special regard to the roles of the Hox genes in the specification of neural lineages with axial-specific architectures, and the roles of other miRNAs on behavior are being systematically tested (Picao-Osorio, 2015).
That no obvious neuro-anatomical changes in miRNA mutant embryos could be detected suggests that these are either very subtle or that the role of miRNA regulation may be primarily behavioral, in the sense of affecting the performance of a correctly wired neural system, rather than developmental, contributing to the development of the network. Given that miR-iab4/iab8 is involved in adult ovary innervation, it seems that miRNAs -- much like ordinary protein-coding genes -- can be involved in several distinct roles within the organism (Picao-Osorio, 2015).
The results of this study contribute to the understanding of how complex innate behaviors are represented in the genetic program. The data lead to a proposal that other miRNAs might also be involved in the control of behavior in Drosophila and other species (Picao-Osorio, 2015).
Double-label RNA FISH and antibody staining was used to determine the relative expression patterns of iab-4 RNA and Ubx RNA/protein accumulation during embryonic development. The iab-4 primary transcript is strongly expressed in the presumptive abdomen, mainly in the progenitors of the second (A2) through seventh (A7) segments (see Cumberledge, 1990). There is also a weak transient stripe of expression in anterior regions. Ubx RNA is distributed in a strong stripe in parasegment 6, but only low levels are seen in regions of the presumptive abdomen containing high levels of iab-4 transcript. No Ubx protein is detected at this early stage, possibly due to the time required to transcribe the entire ~80-kb locus (Ronshaugen, 2005).
During the rapid phase of germband elongation, Ubx protein becomes detectable in the abdomen. At the conclusion of germband elongation, the Ubx and iab-4 patterns are largely complementary in the dorsal ectoderm. During segmentation of the germband, the Ubx protein shows complex modulation in the dorsal ectoderm. There are three apparent levels of Ubx protein distribution in these regions: high, low, and none. An inverse correlation is noted between the levels of Ubx protein and the sites of iab-4 expression. The strongest expression of iab-4 occurs in regions having the lowest accumulation of Ubx protein, whereas intermediate and low levels coincide with sites of diminished Ubx accumulation; there is little or no iab-4 expression in those cells containing the highest levels of Ubx protein. These observations are consistent with the possibility that Ubx protein synthesis might be modulated by one or both iab-4 miRNAs. Direct support for this possibility stems from the analysis of a GFP-Ubx transgene containing the 3' UTR sequence from Ubx. This transgene displays slightly diminished expression in abdominal regions containing high levels of iab-4 transcripts (Ronshaugen, 2005).
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date revised: 26 December 2015
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