miR-124 Regulates Diverse Aspects of Rhythmic Behavior in Drosophila.

Circadian clocks enable organisms to anticipate and adapt to fluctuating environmental conditions. Despite substantial knowledge of central clock machineries, we have less understanding of how the central clock's behavioral outputs are regulated. Here, we identify Drosophila miR-124 as a critical regulator of diurnal activity. During normal light/dark cycles, mir-124 mutants exhibit profoundly abnormal locomotor activity profiles, including loss of anticipatory capacities at morning and evening transitions. Moreover,mir-124 mutants exhibited striking behavioral alterations in constant darkness (DD), including a temporal advance in peak activity. Nevertheless, anatomical and functional tests demonstrate a normal circadian pacemaker in mir-124 mutants, indicating this miRNA regulates clock output. Among the extensive miR-124 target network, heterozygosity for targets in the BMP pathway substantially corrected the evening activity phase shift in DD. Thus, excess BMP signaling drives specific circadian behavioral output defects in mir-124 knock-outs. SIGNIFICANCE STATEMENT: Circadian clocks control rhythmic behaviors of most life-forms. Despite extensive knowledge of the central clock, there is less understanding of how its behavioral outputs are regulated. Here, we identify a conserved neural microRNA as a critical regulator of diurnal behavior. We find Drosophila mir-124 mutants exhibit robust activity abnormalities during normal light/dark cycles and during constant darkness. Nevertheless, as the central pacemaker is functional in these mutants, miR-124 regulates clock output. We provide mechanistic insight by showing deregulation of miR-124 targets in BMP signaling drives specific mir-124 defects. In summary,Drosophila mir-124 mutants reveal post-transcriptional control of circadian activities, and impact of BMP signaling in behavioral output.

A Drosophila genetic screen yields allelic series of core microRNA biogenesis factors and reveals post-developmental roles for microRNAs.

Canonical animal microRNAs (miRNAs) are ∼22-nt regulatory RNAs generated by stepwise cleavage of primary hairpin transcripts by the Drosha and Dicer RNase III enzymes. We performed a genetic screen using an miRNA-repressed reporter in the Drosophila eye and recovered the first reported alleles of fly drosha, an allelic series of its dsRBD partner pasha, and novel alleles of dicer-1. Analysis of drosha mutants provided direct confirmation that mirtrons are independent of this nuclease, as inferred earlier from pasha knockouts. We further used these mutants to demonstrate in vivo cross-regulation of Drosha and Pasha in the intact animal, confirming remarkable conservation of a homeostatic mechanism that aligns their respective levels. Although the loss of core miRNA pathway components is universally lethal in animals, we unexpectedly recovered hypomorphic alleles that gave adult escapers with overtly normal development. However, the mutant photoreceptor neurons exhibited reduced synaptic transmission, without accompanying defects in neuronal development or maintenance. These findings indicate that synaptic function is especially sensitive to optimal miRNA pathway function. These allelic series of miRNA pathway mutants should find broad usage in studies of miRNA biogenesis and biology in the Drosophila system.

A double-negative gene regulatory circuit underlies the virgin behavioral state.

Virgin females of many species conduct distinctive behaviors, compared with post-mated and/or pregnant individuals. In Drosophila, this post-mating switch is initiated by seminal factors, implying that the default female state is virgin. However, we recently showed that loss of miR-iab-4/8-mediated repression of the transcription factor Homothorax (Hth) within the abdominal ventral nerve cord (VNC) causes virgins to execute mated behaviors. Here, we use genomic analysis of mir-iab-4/8 deletion and hth-microRNA (miRNA) binding site mutants (hth[BSmut]) to elucidate doublesex (dsx) as a critical downstream factor. Dsx and Hth proteins are highly complementary in CNS, and Dsx is downregulated in miRNA/hth[BSmut] mutants. Moreover, virgin behavior is highly dose sensitive to developmental dsx function. Strikingly, depletion of Dsx from very restricted abdominal neurons (SAG-1 cells) abrogates female virgin conducts, in favor of mated behaviors. Thus, a double-negative regulatory pathway in the VNC (miR-iab-4/8 ˧ Hth ˧ Dsx) specifies the virgin behavioral state.

miRNAs and Neural Alternative Polyadenylation Specify the Virgin Behavioral State.

How are diverse regulatory strategies integrated to impose appropriately patterned gene expression that underlie in vivo phenotypes? Here, we reveal how coordinated miRNA regulation and neural-specific alternative polyadenylation (APA) of a single locus controls complex behaviors. Our entry was the unexpected observation that deletion of Bithorax complex (BX-C) miRNAs converts virgin female flies into a subjective post-mated behavioral state, normally induced by seminal proteins following copulation. Strikingly, this behavioral switch is directly attributable to misregulation of homothorax (hth). We localize specific CNS abdominal neurons where de-repressed Hth compromises virgin behavior in BX-C miRNA mutants. Moreover, we use genome engineering to demonstrate that precise mutation of hth 3' UTR sites for BX-C miRNAs or deletion of its neural 3' UTR extension containing most of these sites both induce post-mated behaviors in virgins. Thus, facilitation of miRNA-mediated repression by neural APA is required for virgin females to execute behaviors appropriate to their internal state.

Hox miRNA regulation within the Drosophila Bithorax complex: Patterning behavior.

The study of Drosophila Hox genes, located in the Antennapedia complex (ANT-C) and Bithorax complex (BX-C), has provided fundamental insights into mechanisms of how the segments of the animal body plan are specified. Notably, even though the analysis of the BX-C formally began over a century ago, surprises continue to emerge regarding its regulation and function. Even simply the gene content of the BX-C has been regularly revised in past years, especially with regard to non-coding RNAs (ncRNAs), including microRNAs. In this perspective, we review the history of studies of non-coding transcription in the BX-C, and highlight recent studies of its miRNAs that provide new insights into their tissue-specific roles in Hox gene regulation. In particular, we have demonstrated unexpected importance of endogenous BX-C miRNAs to restrict the spatial accumulation of Hox proteins and their TALE cofactors in the ventral nerve cord, and link this to aberrant neural differentiation and reproductive behavior. These findings open new directions on studying Hox miRNA function, and we speculate that further understanding of their roles in insect models may provide new leads for studying the enigmatic biological functions of analogous miRNAs located in vertebrate Hox clusters.

Homeotic function of Drosophila Bithorax-complex miRNAs mediates fertility by restricting multiple Hox genes and TALE cofactors in the CNS.

The Drosophila Bithorax complex (BX-C) Hox cluster contains a bidirectionally transcribed miRNA locus, and a deletion mutant (Δmir) lays no eggs and is completely sterile. We show these miRNAs are expressed and active in distinct spatial registers along the anterior-posterior axis in the CNS. Δmir larvae derepress a network of direct homeobox gene targets in the posterior ventral nerve cord (VNC), including BX-C genes and their TALE cofactors. These are phenotypically critical targets, because sterility of Δmir mutants was substantially rescued by heterozygosity of these genes. The posterior VNC contains Ilp7+ oviduct motoneurons, whose innervation and morphology are defective in Δmir females, and substantially rescued by heterozygosity of Δmir targets, especially within the BX-C. Collectively, we reveal (1) critical roles for Hox miRNAs that determine segment-specific expression of homeotic genes, which are not masked by transcriptional regulation; and (2) that BX-C miRNAs are essential for neural patterning and reproductive behavior.

Function and specificity of Hox genes.

The Hox genes specify different structures along the anteroposterior axis of bilaterians. They code for transcription factors including a conserved domain, the homeodomain, that binds DNA. The specificity of Hox function is determined by each gene controlling the expression of different groups of downstream genes. These can be other transcription factors, elements in signaling pathways or realizator genes that carry out basic cellular functions. In regulating specific targets, the Hox genes interact with members of signaling pathways and with other proteins, thus forming part of gene networks that contribute to the modification of homologous structures or to the creation of new organs.

Polycomb-dependent Ultrabithorax Hox gene silencing induced by high Ultrabithorax levels in Drosophila.

The Ultrabithorax (Ubx) gene of Drosophila specifies the third thoracic and first abdominal segments. Ubx expression is controlled by several mechanisms, including negative regulation by its own product. We show here that if Ubx expression levels are inappropriately elevated, overriding the auto-regulatory control, a permanent repression of Ubx is established. This continuous repression becomes independent of the presence of exogenous Ubx and leads to the paradoxical result that an excess of Ubx results in a phenotype of Ubx loss. The mechanism of permanent repression depends on Polycomb-group genes. Absence of endogenous Ubx transcription when Ubx levels are highly elevated probably activates Polycomb complexes on a Polycomb response element located in the Ubx major intron. This, in turn, brings about permanent repression of Ubx transcription. Similar results are obtained with the gene engrailed, showing that this mechanism of permanent repression may be a general one for genes with negative auto-regulation when levels of expression are transitorily elevated.

The ultrabithorax Hox gene of Drosophila controls haltere size by regulating the Dpp pathway.

The halteres and wings of Drosophila are homologous thoracic appendages, which share common positional information provided by signaling pathways. The activity in the haltere discs of the Ultrabithorax (Ubx) Hox gene establishes the differences between these structures, their different size being an obvious one. We show here that Ubx regulates the activity of the Decapentaplegic (Dpp) signaling pathway at different levels, and that this regulation is instrumental in establishing the size difference. Ubx downregulates dpp transcription and reduces Dpp diffusion by repressing the expression of master of thick veins and division abnormally delayed and by increasing the levels of thick veins, one of the Dpp receptors. Our results suggest that modulation in Dpp expression and spread accounts, in part, for the different size of halteres and wings.