MicroRNA-encoded behavior in Drosophila

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Science  13 Nov 2015:
Vol. 350, Issue 6262, pp. 815-820
DOI: 10.1126/science.aad0217


The relationship between microRNA (miRNA) regulation and the specification of behavior is only beginning to be explored. We found that mutation of a single miRNA locus (miR-iab4/iab8) in Drosophila larvae affects the animal’s capacity to correct its orientation if turned upside down (self-righting). One of the miRNA 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, whereas thermogenetic manipulation of SRN activity results in changes in self-righting behavior. Our work thus reveals a miRNA-encoded behavior and suggests that other miRNAs might also be involved in behavioral control in Drosophila and other species.

MicroRNAs that control behavior

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene activity. They repress expression through complementary base pairing interactions with target messenger RNAs. MiRNAs are involved in regulating many cell and developmental processes. Picao-Osorio et al. find that miRNAs can also control behavior in the fruit fly Drosophila. A specific miRNA locus regulates the self-righting response in larva that ate tipped over onto their backs. The miRNA locus targets a gene required for the normal activity of two neurons involved in the self-righting response.

Science, this issue p. 815

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) (1) have been shown to affect neural differentiation (2, 3), but their roles in the control of behavior are only beginning to be explored.

Previous work in our laboratory focused on the mechanisms and impact of RNA regulation on the expression and neural function of the Drosophila Hox genes (47). These genes encode a family of evolutionarily conserved transcription factors that control specific programs of neural differentiation along the body axis (810), offering an opportunity to investigate how RNA regulation relates to the formation of complex tissues such as the nervous system.

We used the Hox gene system to investigate the roles played by a single miRNA locus (miR-iab4/iab8) (4, 1116) on the specification of the nervous system during early Drosophila development. This miRNA locus controls the embryonic expression of posterior Hox genes (4, 1116). Given that we found no detectable differences 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 (14)] (fig. S3, B to F), we analyzed early larval behavior as a stratagem to probe the functional integrity of the late embryonic nervous system.

Most behaviors in early larva were unaffected by the miRNA mutation (fig. S1 and movies S1 and S2), except self-righting (SR) behavior (Fig. 1, A to C, and movies S3 and S4): miRNA mutant larvae were unable to return to their normal orientation at the same speed as their wild-type counterparts.

Fig. 1 Both removal of miR-iab4/iab8 and overexpression of Ubx disrupt a specific larval locomotor behavior: self-righting (SR).

(A and B) Description of larval SR behavior. (A) Time lapses of larval SR behavior. (Top) Wild-type larvae placed in an inverted position (ventral up), twisted their heads, grabbed the substrate with the mouth hooks, and rolled their bodies onto their ventral surface (dorsal up). (Bottom) In contrast, ΔmiR larvae displayed problems in self-righting their bodies. (B) Diagram of the self-righting behavioral response. (C) Quantification of the time required for the successful completion of the SR behavior (mean ± SEM; n = 27 to 29 larvae per genotype) in two wild-type controls (OR and w1118, light and dark gray, respectively) and ΔmiR larvae (red). (D to F) Quantification of larval behavior in Ubx overexpression lines (UbxM1>Ubx and UbxM3>Ubx). Quantification of (D) number of forward peristaltic waves per minute, (E) larval turning per minute, and (F) time to self-right in wild type (w1118 , gray), ΔmiR (red), and UbxM1>Ubx and UbxM3>Ubx (black) (mean ± SEM; n = 15 to 29 larvae per genotype). A nonparametric Mann-Whitney U test was performed to compare treatments; P > 0.05 (nonsignificant; n.s.); P < 0.001 (***).

By means of selective target overexpression followed by SR phenotype analyses, we identified the Drosophila Hox gene Ultrabithorax (Ubx) (17, 18) as a miRNA target implicated in the genetic control of SR behavior (Fig. 1F). 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 (Fig. 1, D and E). Analysis of Ubx 3′ untranslated region (3′UTR) fluorescent reporter constructs expressed in the Drosophila central nervous system (CNS) (fig. S2) indicates that the interaction between miR-iab4/iab8 and Ubx is direct, which is in line with prior observations in other cellular contexts (1113).

To identify the cellular basis for SR control, we systematically overexpressed Ubx within subpopulations of neurons (fig. S4). Increased levels of Ubx within the pattern of Cha(7.4kb)-Gal4, which largely targets cholinergic sensory and interneurons, phenocopied the miRNA SR anomalies (fig. S4). Further overexpression analysis identified two metameric neurons as the minimal node required for the SR behavior [self-righting node (SRN)] (Fig. 2, A and B).

Fig. 2 miRNA-dependent Ubx regulation in SRN cells underlies SR behavior.

(A) R54F03-GAL4 expression [green fluoprescent protein (GFP), green] in the larval abdominal CNS. Even-skipped protein is in red; Engrailed protein is in blue; and A2, A3, and A4 refer to abdominal segments 2 to 4, respectively. (B) Artificial increase of Ubx expression in two metameric neurons driven by the R54F03 promoter (mean ± SEM; n = 60 larvae per genotype). (C) Artificial decrease of Ubx expression with UbxRNAi within SRN cells in ΔmiR larvae (R54F03-GAL4, ΔmiR/UAS-UbxRNAi, ΔmiR, green) (mean ± SEM; n = 20 to 23 larvae per genotype). (D to I) Conditional increase of Ubx expression during embryonic and early larval development with tub-Gal80ts (Gal80ts represses GAL4 activity at 18°C) within SRN cells: R54F03 > Ubx, tub-GAL80ts (UAS-Ubx/+ ; R54F03/tub-Gal80ts). Controlled increase of Ubx expression in SRN cells in early larvae [(D) and (E); mean ± SEM; n = 20 larvae per genotype] and from mid-embryogenesis to early larvae [(H) and (I); mean ± SEM; n = 15 larvae per genotype]. [(F) and (G)] Repressed increase of Ubx expression in SRN cells throughout embryogenesis and early larvae (mean ± SEM; n = 15 larvae per genotype). A nonparametric Mann-Whitney U test was performed to compare treatments; P > 0.05 (nonsignificant, n.s.); ***P < 0.001.

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 (Fig. 3, A to C). Second, in the context of miRNA mutation, Ubx protein expression is increased within the SRN (Fig. 3, D to F). 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 (Fig. 2C).

Fig. 3 Regulation of Ubx protein expression in SRN cells by miR-iab4/iab8.

(A) Wild-type expression of precursor miR-iab4 transcripts [RNA–fluorescence in situ hybridization (RNA-FISH), purple] in SRN cells (R54F03>GFP, green) of the ventral nerve cord (VNC) of first-instar Drosophila larvae. (B) Wild-type expression of precursor miR-iab8 transcripts (RNA-FISH, blue) in SRN cells (R54F03>GFP, green) of the VNC of first-instar Drosophila larvae. (C) Percentage of SRN cells expressing miR-iab4 (purple, square) and miR-iab8 (blue, triangle) precursors across A1 to A6 (n = 10 larvae). (D and E) Ubx protein expression (red) in SRN cells of wild-type (D) and ΔmiR (E) first-instar larvae VNCs. (F) Quantification of Ubx protein expression ratio of ΔmiR over wild type within the SRN cells (red) by fluorescent intensity (n = 8 larvae per genotype; arbitrary units, a.u.). (G) Diagram of a subregion of the bithorax complex based on (14) showing iab-4 (purple) and iab-8 (blue) noncoding RNAs (ncRNAs), and rearrangement breakpoints affecting miR-iab-4 (iab-3277, purple) and miR-iab-8 (iab-5105 and iab-7MX2, blue). (H) Genetic complementation tests to determine the involvement of miR-iab4 or miR-iab8 in SR behavior by using trans-heterozygote larvae for ΔmiR and different chromosomal rearrangement breakpoints that disrupt the bithorax complex (mean ± SEM; n = 17 to 20 larvae per genotype). A nonparametric Mann-Whitney U test was performed to compare treatments; P > 0.05 (nonsignificant; n.s.); *P < 0.05; **P < 0.01; ***P < 0.001.

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 (19, 20)] show no significant differences in total numbers of SRN cells (fig. S5B) or in SRN cell body size (fig. S5C); furthermore, analysis of wt, ΔmiR, and R54503>Ubx show indistinguishable SRN-projection patterns (fig. S5, D and E). Second, Gal-80ts–mediated conditional expression experiments show that SRN-specific Ubx overexpression after embryogenesis is sufficient to trigger the SR behavior (Fig. 2, D and E).

The results presented above 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, we used genetically encoded calcium sensors [GCaMP6 (21)] specifically expressed in SRN cells and tracked down spontaneous profiles of neural activity. SRN cells in miRNA mutants produce activity traces that are significantly different from those observed in wild-type SRN cells (Fig. 4, B and C, and fig. S6A). Quantification of maximal amplitude and proportion of active cells in each genotype also reveal significant differences (Fig. 4D and fig. S6B) in SRN function across the genotypes, but no change in cell viability is observed (fig. S6C). Neural activity differences across genotypes are significant within regions of expression of miR-iab4 (Fig. 4E), 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 (14, 15, 22, 23) strongly suggests that miR-iab4 is the key regulator of SR (Fig. 3, G and H).

Fig. 4 ΔmiR mutants have abnormal patterns of neural activity in SRN cells.

(A) Schematic of the larval CNS expressing GCaMP6m in SRN cells (R54F03> GCaMP6m, green) imaged in a two-photon microscope. (B) Examples of spontaneous activity recorded over 10 min from wild type (WT: UAS-GCaMP6m/+; R54F03-GAL4/+) and (C) ΔmiR mutants (UAS-GCaMP6m/+; R54F03-GAL4, ΔmiR/ΔmiR) in SRN cells. (D) Maximum amplitude of spontaneous activity in SRN cells: WT (median ΔF/F = 1.91; n = 120 SRN cells) and in ΔmiR mutants (median ΔF/F = 1.27; n = 115 SRN cells) (**P < 0.01, Mann-Whitney U test). (E) Expression pattern of miR-iab4 (purple) and 4′,6-diamidino-2-phenylindole (DAPI, blue) in the VNC of a freshly hatched larva (left). Median ΔF/F in SRN cells of WT (black line) and ΔmiR (red line) larval VNCs, and relative expression of miR-iab4 (purple) along the anterior-posterior (A-P) axis. Median ΔF/F of WT (median of 2.132, n = 73 SRN cells) and ΔmiR (median of 1.122, n = 68 SRN cells) in regions of high miR-iab4 expression (**P < 0.01, Mann-Whitney U test). Regions of low miR-iab4 expression have a median ΔF/F of 1.763 in WT (n = 47 SRN cells) and 1.749 (n = 47 SRN cells) in ΔmiR specimens (n.s., P > 0.05; Mann-Whitney U test). (F and G) Thermogenetic manipulation of neural activity in SRN cells. Activation [(F), R54F03>dTrpA1] and inhibition [(G), R54F03>shits] of SRN neural activity (mean ± SEM; n = 17 larvae per genotype, ***P < 0.0001, Mann-Whitney U test) [29°C (green) for activation (H) and 36°C (orange) for inhibition (I)]. (H) Wild-type motor axonal projections of SRN cells (UAS-myr::GFP/UAS-myr::GFP; R54F03-GAL4/R54F03-GAL4, green) into muscles (phalloidin, red) lateral transverse 1 and 2 (LT1 and LT2) in late embryos (stage 17) (Fasciclin II, FASII, blue). (I) Diagram of SRN cells projecting to the LT1 and LT2 muscles. (J) A model that summarizes the data reported in this study. Mutation of miR-iab4 (left) leads to Ubx derepression in the SRN node, affecting SRN neural activity patterns and triggering an anomalous SR behavior (right).

To demonstrate that the changes in SRN neural activity were causal to SR behavior, we artificially activated (Fig. 4F) or inhibited (Fig. 4G) SRN cells (24, 25) and showed that this triggered 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. We developed an optogenetic system in which we activated SRN cells 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 (fig. S7 and movie S5). Neither parental line R54F03-Gal4 nor UAS-Ch2R showed similar reactions to stimulation, confirming the specificity of this effect (fig. S7 and movies S6 and S7).

To study the links between SRN neurons and the SR movement, we labeled SRN projections with myr-GFP and discovered that SRN cells innervate two of the lateral transverse (LT) muscles and that they can be colabeled antibodies against Fasciclin 2 (Fas2) (Fig. 4H), demonstrating these to be motorneurons. LT muscles are innervated by Bar-H1+ motorneurons (fig. S8A), so we used Bar-H1-Gal4 as a second driver to demonstrate that appropriate Ubx levels in these cells are required for normal SR behavior (fig. S8B), establishing the SRN cells as the LT-MNs.

We have 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. Our 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; we are currently investigating this possibility, with special regard to the roles of the Hox genes in the specification of neural lineages with axial-specific architectures, and systematically testing the roles of other miRNAs on behavior.

That we could not detect any obvious neuro-anatomical changes in miRNA mutant embryos 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 (26). Given that miR-iab4/iab8 is involved in adult ovary innervation (16), it seems that miRNAs—much like ordinary protein-coding genes—can be involved in several distinct roles within the organism.

The results of this study contribute to the understanding of how complex innate behaviors are represented in the genetic program. Our data lead us to propose that other miRNAs might also be involved in the control of behavior in Drosophila and other species.

Supplementary Materials

Materials and Methods

Figs. S1 to S11

References (2748)

Movies S1 to S7

References and Notes

  1. Acknowledgments: We thank L. Lagnado for his support to this project and S. Pinho and P. Reed for technical assistance. We also thank R. White for antibodies; Welcome Bender, E. Sánchez-Herrero, and the Bloomington Stock Centre for Drosophila stocks; and P. Patraquim for bioinformatic support. This paper is dedicated to the memory of Amalia Lamuedra de Alonso for her devoted support to this work. This research was funded by a Wellcome Trust Investigator Award to C.R.A. [WT grant 098410/Z/12/Z] and a Ph.D. studentship to J.P.O. by Fundação para a Ciência e a Tecnologia (Portugal) [FCT grant SFRH/BD/63312/2009]. J.B. is funded by Sir Henry Dale Fellowship (Wellcome Trust and the Royal Society) Grant 105568/Z/14/Z, and M.L. was supported by grants from the Biotechnology and Biological Sciences Research Council (UK) (BB/I022414/1) and the Wellcome Trust (092986/Z).
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