Following the general rule of posterior prevalence, by which posterior homeotic genes repress the expression of the more anterior ones (Gonzalez-Reyes et al. 1990, Bachiller et al. 1994), abd-A has been long thought to be repressed by Abd-B in PS13 (Karch et al.
1990, Macias et al. 1994).
Posterior prevalence, despite being a general rule, has proven to be broken in some cases.
In the genital disc, abd-A and Abd-B not only show co-expression, but both are necessary for the correct development of adult genitalia (Foronda et al. 2006). There is a similar situation in the abdominal histoblasts during pupation, in which the co-expression between abd-A and Abd-B is necessary for the development of the epidermis (Singh and Mishra 2014). Finally, our laboratory has shown that Abd-B is not only co-expressed with abd-A in some cells of the embryonic central nervous system, but that it is also unable to repress it. Even more, the CNS cells that express higher amounts of ABD-A express also high amounts of ABD-B, hinting that this co-expression could be important in the developing nervous system (Gummalla et al. 2012).
Interestingly, the expression of abd-A in the CNS of Abd-B- mutants, is always restricted to PS7-12, being absent in PS13-14.
Extensive studies on abd-A regulation in the CNS shed light into its repression in PS13 of the embryonic central nervous system, showing not only that this repression is independent of Abd-B expression, but introducing as a new actor in this regulation the iab-8 ncRNA. This noncoding RNA represses abd-A expression in PS13 of the CNS, via two mechanisms: the mir-iab-8 microRNA and a cis-acting mechanism proposed to be transcriptional interference (Gummalla et al. 2012, Gummalla et al. 2014).
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As mentioned above, the promoter of the iab-8 ncRNA lies very close to the Fab-8 boundary that delimits iab-7 and iab-8. It turned out that one of our Fab-8 boundary deletions also remove the promoter of the iab-8 ncRNA (Fab-864). As this deletion has no consequence on Abd-B regulation in PS13, we often use Fab-864 as source of mutation lacking the iab-8 ncRNA. The deletion of the promoter of the iab-8 ncRNA in Fab-864 results into the complete derepression of abd-A in this parasegment of the CNS (Gummalla et al. 2012, Gummalla et al. 2014). This demonstrates that the iab-8 ncRNA is the only repressor of abd-A in this tissue (Figure VI).
Figure VI. abd-A repression in PS13 of the CNS disappears in absence of the iab-8 ncRNA
Dissected embryonic central nervous systems immunostained against ABD-A (red) and ABD-B (green) in WT and Fab-864 embryos. Modified from (Gummalla et al. 2014).
36 The mir-iab-8 microRNA
As described in the previous section, the transcriptional unit of the iab-8 ncRNA contains a hairpin that is responsible for the formation of the mir-iab-8 microRNA. The importance of this regulatory element is not small, as the ΔmiR mutation, which consists on a surgical deletion of the microRNA hairpin, cause a striking phenotype consisting in complete sterility. This phenotype must be related to neuromuscular defects, as ΔmiR males cannot bend their abdomen for mating and ΔmiR females are unable to lay eggs, due to defective contraction of the oviduct (Bender 2008).
One striking feature of the mir-iab-8 repression over abd-A is its exceptional strength.
For example, GAL4-induced overexpression of mir-iab-8 in the genital disc leads to complete repression of abd-A in this tissue (Tyler et al. 2008). In addition, the failure of transcriptional interference in the CNS, which leaves the mir-iab-8 as the sole mechanism of repression of abd-A, has as an effect the derepression of abd-A in only a few cells, suggesting that mir-iab-8 is able to completely repress this target in most cells of PS13 of the CNS (Gummalla et al. 2012) This constitutes an exception to the general rule action of microRNAs, which states that most microRNAs expressed at endogenous levels simply tune the levels of expression of their target genes and, in the cases where this repression is stronger, only causes a maximum of 80% of repression (Kozomara et al.
2014)
The production of mir-iab-8 is not the only function of the iab-8 ncRNA
The discovery of the breaking of posterior prevalence of Abd-B over abd-A in the embryonic central nervous system, and the characterization of the iab-8 ncRNA as repressor of abd-A in PS13 of this tissue, led to an exhaustive study on abd-A expression in this tissue in different mutant contexts (Gummalla 2011, Gummalla et al. 2012, Gummalla et al. 2014).
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One of the most interesting results described in this study was the different degree of abd-A derepression in PS13 of the CNS showed by embryos homozygous for different chromosomal rearrangements, which disrupted the transcriptional region of the iab-8 ncRNA at different locations (Figure VII):
Figure VII. The disruption of the iab-8 ncRNA transcriptional unit at various locations has different effects over abd-A expression in PS13 of the CNS
Dissected embryonic central nervous systems immunostained against ABD-A (red) and EN (green) in WT embryos and in embryos homozygous for the chromosomal rearrangements iab-333, iab-35022, iab-4186, iab-7SGA62; and for the deletion of the mir-iab-8 hairpin (ΔmiR). EN marks the anterior border of each parasegment. The arrows in the graph show the points in which the transcriptional unit of the iab-8 ncRNA is disrupted in each chromosomal break. Red arrows mark chromosomal breaks that allow the formation of the mir-iab-8 microRNA, and blue arrows indicate those who prevent it.
Modified from (Gummalla et al. 2012).
Breakpoints occuring upstream from the mir-iab-8 hairpin (iab-7SGA62, iab-4186, shown in blue) prevent the formation of this microRNA, and also prevent the transcription of the iab-8 ncRNA until its terminal exon. Homozygous embryos for these chromosomal rearrangements show a complete derepression of abd-A in PS13 of the CNS.
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Breakpoints occuring downstream from the hairpin (iab-333, iab-35022, shown in red), allow the transcription over the microRNA hairpin (therefore producing mir-iab-8) but prevent the progression of the RNA polymerase II towards exon 8. These embryos show abd-A derepression only in a few cells of the CNS. This does not come as a surprise, as the mir-iab-8 microRNA is produced in this case. However, the slight derepression means that the microRNA is not able to totally repress abd-A in PS13. Therefore, the authors suggested the existence of a second repressive mechanism. This claim found support in the finding that, in ΔmiR homozygous embryos, which do not produce the mir-iab-8 microRNA but have the rest of the iab-8 ncRNA sequence intact, abd-A is still quite repressed in PS13, appearing in only a few neurons (Figure VII, ΔmiRNA).
The locus responsible for the second repressive mechanism over abd-A resides downstream from the microRNA hairpin, probably near the terminal exon, which ends only 1Kb upstream from the promoter of abd-A. A simple and clever experiment suggested that this second repressive mechanism was only able to act in cis- over abd-A (Figure VIII).
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Figure VIII. The microRNA-independent repression exerted by the iab-8 ncRNA over abd-A acts only in cis-
Dissected embryonic central nervous systems immunostained against ABD-A in ΔmirR/+
and ΔmiR/mfs5649 embryos. The scheme in the lower part of the figure represents the potential repression exerted by the microRNA-independent function of the iab-8 ncRNA over the other chromosome, if this mechanism was able to act in trans-.
Adapted from (Gummalla et al. 2012).
When the microRNA deletion ΔmiR is placed over a deficiency for the whole iab-8 ncRNA (like in this case mfs5649, a P element insertion that blocks the transcription of the iab-8 ncRNA very close to its promoter), only one chromosome (the mfs5649) lacks the secondary repressive function. If the secondary mechanism was able to act in trans, the repression exerted by the ΔmiR chromosome should be sufficient to repress both.
However, this is not the case, and abd-A becomes totally derepressed in PS13 of the embryonic CNS, allowing the authors to discard the possibility of a diffusible factor (micropeptide or microRNA) being the responsible for the secondary repression mechanism.
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The fact that the secondary repressive mechanism needs transcription of the iab-8 ncRNA to arrive near the promoter of abd-A, combined with the fact that it seems to be a cis-acting mechanism, led the authors to propose transcriptional interference as the most likely model for the repression of abd-A.
Evolutionary conservation of the iab-8 ncRNA
The mir-iab-4/mir-iab-8 bidirectionally transcribed hairpin, along with the binding sites for its corresponding microRNAs in the 3'UTR of their main target genes, are conserved in almost all Arthropods (Ronshaugen et al. 2005, Tyler et al. 2008, Miura et al. 2011, Hui et al. 2013). There is only one described case in which this microRNA has not been found, the chelicerate Tetranynchus urticae. Strikingly, this unique loss of the otherwise extensively conserved mir-iab-8 microRNA is in correlation with the simultaneous loss of abd-A in this species. This could suggest that the ancestral function of this microRNA could have been the repression of abd-A (Pace et al. 2016).
Our laboratory has shown that the iab-8 ncRNA transcript arrives near the promoter of abd-A in at least two other Drosophila species: D. pseudoobscura and D. virilis. In these species, its expression is also restricted to PS13-14, and this gene shows a similar tissue specificity at the end of embryonic development (Gummalla et al. 2012). More recently, it has been discovered that the mir-iab-8 hairpin is transcribed with the same expression pattern in the beetle Tribolium castaneum (Hui et al. 2013), and a transcript that seems to correspond to the iab-8 ncRNA has been identified by RT-PCR in the silkworm Bombyx mori (Wang et al. 2019).
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