Abstract
How different organs in the body sense growth perturbations in distant tissues to coordinate their size during development is poorly understood. Here, we mutated an invertebrate orphan relaxin receptor, the Drosophila Lgr3, and found body asymmetries similar to those found in insulin/relaxin-like peptide 8 (dilp8) mutants, which fail to coordinate growth with developmental timing. Indeed, mutation or RNAi against Lgr3 suppresses the delay in pupariation induced by imaginal disc growth perturbation or ectopic Dilp8 expression. By fluorescently-tagging the endogenous Lgr3 protein and performing CNS-specific RNAi, we find that Lgr3 is expressed and required in a novel subset of CNS neurons to transmit the peripheral tissue stress signal, Dilp8, to the neuroendocrine centers controlling developmental timing. Our work sheds new light on the function and evolution of relaxin receptors and reveals a novel neuroendocrine circuit responsive to growth aberrations.
Main text
How different organs in the body sense growth perturbations in distant tissues to coordinate their size and differentiation status during development is poorly understood1,2. We have previously discovered a hormone in Drosophila, the insulin/relaxin-like peptide Dilp8, which ensures organ and body size coordination3. In developing larvae, Dilp8 is produced and secreted from abnormally-growing imaginal discs. Its activity transiently delays the onset of metamorphosis by inhibiting the biosynthesis of the major insect molting hormone ecdysone by the prothoracic gland, a part of a compound endocrine structure called the ring gland3,4 (Fig. 1A). Loss of dilp8 uncouples the endocrine communication between imaginal discs and the prothoracic gland, making dilp8 mutants susceptible to uncoordinated disc growth. This results in an increase in random deviations from bilateral symmetry [fluctuating asymmetry (FA)], measurable at the population level3. These findings have placed Dilp8 as a central player in the interorgan communication system that mediates plasticity to promote developmental stability in Drosophila. However, which molecule(s) and tissue(s) sense and/or transmit this abnormal growth signal to the prothoracic gland are unknown.
Type C1 Leucine-rich repeat-containing G-protein coupled receptors (Lgrs) are a highly conserved protein family in metazoans that act as receptors for insulin-like peptides of the relaxin subfamily in vertebrates, where they play diverse roles in tissue homeostasis and remodeling, behavior and reproduction5. In invertebrates, however, they are considered orphan receptors and their biological function is a mystery because invertebrates are thought to lack bona fide relaxin peptide homologues5-7. The Drosophila genome encodes two orphan receptors, Lgr3 and Lgr4, with clear homologies to vertebrate relaxin receptors [∼45% and ∼40% sequence similarity to human, RXFP1/2, respectively6,7]. To start untangling the function of these relaxin receptors, we remobilized an MB Minos element8 to generate a mutant for the Drosophila Lgr3 (Fig. 1B). The obtained imprecise excision allele was named Lgr3ag1. Three precise excisions (Lgr3+/+) were also retained and used as genetic background controls. The Lgr3ag1 imprecise excision (Supplementary Fig. 1A-D) consists of a 3.8-kb deletion that completely removes exon 8 and partially removes exon 9 (Fig. 1B). Reverse-transcriptase polymerase chain reaction assays followed by Sanger sequencing determined that the Lgr3ag1 deletion leads to readthrough from exon 7 directly into intron 7 and usage of an aberrant splicing donor within intron 7 directly into exon 9 (Fig. 1B and Supplementary Fig. 2A-D). The resulting transcript therefore has an intron-encoded premature termination codon (PTC) that truncates the Lgr3 protein one aminoacid after D326 (Fig. 1C). We conclude that Lgr3ag1 encodes a severely compromised protein that is unlikely to bind ligand or signal due to a truncated ligand-binding LRR domain5, and absence of the seven transmembrane (7TM) domains and G protein coupling Carboxy terminus (Fig. 1C).
Lgr3 couples growth to developmental timing
We noticed increased FA in Lgr3ag1 adult wings. FA indexes (FAi) are increased by an order of ∼3 when compared to their Lgr3+/+ controls [p = 0.0028, F-test for wing area FAi (Fig. 1D)]. This phenotype is indicative of uncoordinated imaginal disc growth during the larval stage, when wing size is determined, and is reminiscent of the increased FA phenotype of animals lacking the insulin/relaxin-like peptide Dilp83. We thus hypothesized that Lgr3 and Dilp8 act on the same pathway to promote developmental stability by coupling imaginal disc growth to developmental timing.
To test the hypothesis that Lgr3 and Dilp8 are in the same pathway, we asked the question whether Lgr3 mutants show a similar defect as dilp8 mutants in the ability to delay the onset of metamorphosis in response to induced abnormal tissue growth3,4. We did this by inducing tissue damage in an Lgr3ag1 background and carried out pupariation timing assays. Tissue damage was induced by placing the proapoptotic gene reaper (rpr) under control of the wing-pouch Beadex-Gal4 (Bx>)3,9, a combination that effectively caused a 72-h (median) delay in pupariation in control Lgr3+/+ animals (Fig. 2A). Strikingly, this delay was reduced by 50 h in Lgr3ag1 mutants [∼80%, p < 0.01, post-hoc test after Conover for Kruskal-Wallis test (hereafter, Conover post-hoc test); Fig. 2A]. The extent of this rescue is in line with previously published results using dilp8 mutants3,4. This result demonstrates that Lgr3, like Dilp8, is critical to mediate the communication between abnormally growing imaginal discs and the prothoracic gland, strongly indicating that Lgr3 and Dilp8 act on the same pathway.
Lgr3 acts in the Dilp8 pathway
To verify whether the Dilp8 activity is Lgr3 dependent, we tested if Lgr3 was necessary for the developmental delay produced by ectopic Dilp8 expression in the absence of tissue growth abnormalities3,4. The delay in development induced by constitutive expression of a UASP-dilp8::3xFLAG (UAS-dilp8a) transgene under the control of the ubiquitous driver [using armadillo-Gal4 (arm>)] was suppressed in larvae homozygous for Lgr3ag1 or trans-heterozygous for Lgr3ag1 and a deficiency that completely uncovers the Lgr3 locus (Lgr3Df(3)BSC321) (p < 0.01, Conover post-hoc test; Fig. 2B). These results show that animals lacking the Drosophila relaxin receptor Lgr3 are insensitive to ectopically produced Dilp8. Furthermore, as the suppression phenotype of ag1 is indistinguishable from the Df allele, the results bring further evidence that ag1 is a strong loss-of-function allele. To test whether Lgr3 activity is required for the dilp8-dependent delay in a completely independent experimental setting, we induced a developmental delay using two previously-reported dilp8 transgenes [UAST-dilp8::3xFLAG (UAS-dilp8b and c)]3 under the control of a ubiquitous driver, tubulin-Gal4 (tub>), and reduced Lgr3 activity by concomitant RNAi knockdown using a short hairpin [TRiP VALIUM22 (Lgr3-IR-V22)] line10, which reduces the steady-state Lgr3 mRNA levels by ∼85% (p=0.043, Student’s t-test) (Supplementary Fig. 3A). Coherent with the mutant analyses, RNAi against Lgr3 completely suppressed the dilp8-dependent delay (Fig. 2C). A second RNAi line producing a long hairpin against Lgr3 [TRiP VALIUM10 (Lgr3-IR-V10)]11 also rescued the dilp8-dependent delay, albeit to a lesser extent than Lgr3-IR-V22 (Supplementary Fig. 3B). This partial rescue was proportional to a weaker reduction in Lgr3 mRNA levels (∼50%) than Lgr3-IR-V22 (Supplementary Fig. 3A), suggesting that the Dilp8 delay is sensitive to Lgr3 dosage. The Lgr3-IR-V22 RNAi transgene was hereafter used in further experiments, as it phenocopies the Lgr3 mutant phenotype best and gives the strongest RNAi reduction. Together with the Lgr3 mutation analyses, the Lgr3 RNAi experiments strongly place Lgr3 in the Dilp8-dependent developmental delay pathway.
Lgr3 is expressed in the CNS
To gain insight into the tissue and cellular expression pattern of Lgr3 at the protein level, we used CRISPR/Cas9 mediated homologous repair12,13 to tag the endogenous Lgr3 protein at its Amino-terminus with superfolder green fluorescent protein (sfGFP14; Fig. 3A-B and Supplementary Fig. 4). We obtained one allele, Lgr3ag5, hereafter named sfGFP::Lgr3, which contained an intact Lgr3 coding sequence downstream of the sfGFP insertion (Supplementary Fig. 4). sfGFP::Lgr3 labels ∼180 CNS cell bodies, consisting of ∼40 cell bodies in the brain proper and ∼140 in the ventral nerve cord (VNC) of 3rd instar larvae CNS (Fig. 3C). No other larval tissue apart from the CNS had detectable fluorescence or anti-GFP staining. This rules out the possibility that Lgr3 cell-autonomously controls ecdysone biosynthesis in the prothoracic gland downstream of Dilp8. The effectiveness of sfGFP::Lgr3 as a protein translation reporter was further confirmed by analyzing alleles with premature termination codon-inducing (PTC+) indels in the Lgr3 coding region following the inserted sfGFP sequence (Supplementary Fig. 4) or by using RNAi against endogenous Lgr3 (Fig. 3C). Results show that either PTCs or RNAi against Lgr3 effectively reduced sfGFP::Lgr3 expression to undetectable levels (Fig. 3C, right panels), demonstrating the reliability of the sfGFP::Lgr3 endogenous protein reporter and further certifying the effectiveness of the Lgr3 RNAi. Importantly, PTC+, but not PTC-alleles suppressed the delay induced either by raising the larvae in the presence of the genotoxic agent ethylmethanosulfonate (EMS), which induces apoptosis, tissue damage/regeneration, an imaginal-disc specific Dilp8 upregulation and consequentially a robust delay in the onset of metamorphosis3 (Fig. 3D) or by ectopically expressing Dilp8 (Supplementary Fig. 5). These results confirm that the PTC+ alleles are loss-of-function alleles and demonstrate that the PTC-sfGFP::Lgr3 ag5 allele encodes a functional receptor. Together, these results strongly suggest that the Lgr3 protein acts in a subpopulation of CNS neurons.
Critically, we were also unable to detect sfGFP::Lgr3 expression in neurons directly innervating the ring gland (Fig. 3C), ruling out the possibility that Lgr3 neurons control developmental timing by direct cellular contact or synapsis with the ring gland, such as through PTTH-producing neurons15, the insulin producing cells (IPCs)16, or subesophageal serotonergic neurons17, all of which have been shown to modulate developmental timing. This scenario suggests that the Lgr3-positive neurons represent a novel distinct cellular population that has not been previously linked to growth and developmental timing control. To gain further insight into how Lgr3 neurons transmit the Dilp8 signal to the prothoracic gland, we looked into the neuroanatomy and neurotransmitter profile of sfGFP::Lgr3 neurons in more detail. Notably, sfGFP::Lgr3 expression was not homogeneous in the ∼180 CNS cell bodies. It was most strongly expressed in a single pair of neurons in the very tip of the VNC, in a single dorso-ventral pair of midline neurons located deep in the thoracic segment of the VNC (hereafter abbreviated to MIL neurons, for midline internal Lgr3 neurons), and in a pair of bilateral neurons localized in the anterior part of the pars intercerebralis (hereafter abbreviated to PIL neurons, for pars intercerebralis Lgr3 neurons) (Fig. 3C, arrows and 3E) and (Fig. 3C, arrows and 3f). All three neuronal populations express sfGFP::Lgr3 and present their characteristic neuroanatomy already at the L1 stage (Supplementary Fig. 6). Co-staining of sfGFP::Lgr3 with choline acetyltransferase (Cha)-Gal4 driving a UAS-myr::tdTomato reporter indicates that many Lgr3 positive neurons are cholinergic, including all three major neuronal populations (PILs, MILs and the pair of distal VNC neurons (Supplementary Fig. 7). In contrast, co-staining with glutamic acid decarboxylase (Gad1)-Gal4 and Vesicular glutamate transporter promoter (VGlut)-Gal4, which drive expression in GABAergic and glutamatergic neurons, respectively, label only very faintly the PIL neurons and the pair of distal VNC neurons and show no detectable staining in MIL neurons (Supplementary Fig. 8). While MIL neurons project their neurites ventrally and then anteriorly towards the brain, branching close to the base of the brain (Fig. 3E), the PIL neurons extend a single neurite centripetally into the neuropil that branches dorsally into highly arborized termini and ventrally into a slightly less arborized projection that proceeds towards the subesophageal region with a yet undetermined terminal branch (Fig. 3F). We noticed that the proximal arborizations of PIL neurons project to a region of the pars intercerebralis known to harbor the IPCs and anterior neurites from Dilp7-producing dorsal medial (DMA1)18 neurons projecting from the VNC. To verify this assumption, we co-stained sfGFP::Lgr3 brains with anti-Dilp5 or anti-Dilp7 as markers for IPCs or DMA1 neuron projections, respectively. Our results show that PIL neuron proximal arborizations project towards the nearby IPC-bodies reaching them from immediately underneath, while the IPCs neurites extend through the PIL neuron arborization (Fig. 3G). PIL neurons also show an intimate association with the axonal projections of the Dilp7-producing DMA1 neurons (Fig. 3H). These results provide anatomical and possible functional context for an anterograde neuronal input for PIL neurons (considering that proximal branches are typically dendritic)19.
Lgr3 acts in the CNS to delay development
The expression pattern of the sfGFP::Lgr3 reporter suggests that disrupting receptor function exclusively in the CNS should be sufficient to suppress the Dilp8-dependent delay in the onset of metamorphosis. To test this, we crossed the Lgr3-IR-V22 RNAi line to the panneuronal CNS driver elav-Gal4 (elav>) and performed EMS assays (Fig. 4A). Reducing Lgr3 function with RNAi in the nervous system using elav> leads to complete suppression of the EMS-induced delay, as also observed with a ubiquitous knockdown [using armadillo-Gal4 (arm>)] (Fig. 4A). The neuronal requirement of Lgr3 was further confirmed by knockdown of Lgr3 in elav cells in a context of ectopic expression of dilp8 (Supplementary Fig. 9A), and by rescuing neuronal expression of Lgr3 in an arm>dilp8 Lgr3-IR-V22 context by inhibiting Lgr3 RNAi exclusively in the CNS by expressing the Gal4 inhibitor, Gal80, under the control of the elav enhancer (elav-Gal80; Supplementary Fig. 9B). As expected from our neuroanatomical and neurotransmitter biosynthesis expression pattern studies, knockdown of Lgr3 in cholinergic neurons using Cha-Gal4 also significantly suppressed the EMS-induced delay (Fig. 4B). In contrast, knockdown of Lgr3 in the ring gland using phantom (phm)-Gal4 line did not lead to a significant rescue of the EMS-induced delay (Fig. 4C). We conclude that Lgr3 is required in the CNS, likely in one or more of the ∼180 Lgr3-positive cholinergic neurons described above, to convey the Dilp8-dependent developmental delay.
If, as described above, neuronal Lgr3 is required to convey the Dilp8-dependent signal that promotes imaginal disc size stability by providing extra developmental time in the presence of disc growth problems, then the removal of Lgr3 only in the CNS should also lead to an increased FAi, similarly to the increased FAi found in animals carrying mutations in Lgr3 (Fig. 1D) or dilp8[3]. Consistent with this hypothesis, we find that FAi is increased by a factor of ∼4 in elav>Lgr3-IR-RNAi compared to controls (p < 0.001, in both F tests) (Fig. 4D). These results provide further evidence that neuronal Lgr3 is critical in the pathway controlling developmental stability.
According to heterologous studies in human cell lines in vitro7, Lgr3 is a constitutively active receptor. If this were true in vivo and if Dilp8 activity would inhibit Lgr3 activity, we would expect that by removing Lgr3 activity we would see a delay, which is not the case (see controls in Fig. 2A, Fig. 3D and Fig. 4A). To look at the effect of removing Lgr3 activity on pupariation timing at an increased resolution, we resynchronized larvae at the second to the third instar molt and confirmed that Lgr3 mutants do not have a delayed development, but rather pupariate ∼4 h earlier than controls, independent of their genetic background (Supplementary Fig. 10). Even though our loss-of-function studies do not rule out constitutive activity, they are more consistent with a scenario where Lgr3 is activated either by Dilp8 or by other downstream Dilp8-dependent signals. We thus hypothesized increased Lgr3 levels would not lead to a delay. Alternatively, if Lgr3 were somehow constitutively active, it would delay pupariation timing if ectopically expressed. To conduct this experiment, we constructed a UASP-Lgr3 transgene and expressed it in the CNS under the control of elav>. We find that the pupariation time profile of elav>Lgr3 animals is indistinguishable from controls (Fig. 4E). Control experiments suggest that the UASP-Lgr3 transgene carries functional Lgr3 activity, as it rescues the FAi of ag1 animals when driven by elav> (Fig. 4F). These results argue against a simple explanation where Lgr3 is constitutively active7. Instead, they are in line with our findings that, in the absence of tissue growth aberrations or ectopic Dilp8 expression, Lgr3 activity does not have a major impact on timing the onset of metamorphosis. These results are coherent with a model where Lgr3 is activated either directly or indirectly by the Dilp8 signal and hint towards the existence of a dedicated tissue stress-sensing pathway in Drosophila.
Lgr3 neurons relay the Dilp8 delay signal
To further narrow down the identity of the neurons requiring Lgr3, we tested the ability of two Janelia Gal4 lines20 carrying regulatory regions of the Lgr3 locus, GMR17G11-Gal4 (GMR17G11>) and GMR19B09-Gal4 (GMR19B09>) (Fig. 5A), selected based on their larval brain expression patterns21, to suppress the EMS-induced developmental delay when driving Lgr3-IR-V22. While GMR17G11>Lgr3-IR-V22 had no effect on the EMS-delay (Supplementary Fig. 11), the GMR19B09>Lgr3-IR-V22 condition significantly reduced the 31.6-h EMS-dependent delay in pupariation time to 15.7 h (∼50% reduction, p < 0.01, Conover post-hoc test; Fig. 5B). We conclude that the subset of neurons expressing GMR19B09> is critical for the Dilp8 and Lgr3-dependent coupling of growth and developmental timing. Silencing of these same neurons by expression the inward rectifying K+ channel (Kir2.1) under the control of the GMR19B09> driver throughout embryonic and larval development was compatible with developmental progression and led to a significant suppression of the EMS-dependent delay (p < 0.01, Conover post-hoc test (Fig. 5C). These results strongly suggest that the activity of GMR19B09> neurons dictates the central Dilp8 and Lgr3-dependent response to tissue damage. To further characterize the neuroanatomy of GMR19B09> cells, we placed this driver together with a UAS-myr::tdTomato reporter transgene in a sfGFP::Lgr3 background and carried out immunofluorescence assays of dissected CNSs. GMR19B09> drives expression in ∼270 neurons, ∼30 of which populate each brain hemisphere21. We find sfGFP::Lgr3 expression in ∼10 GMR19B09>myr::tdTomato-labeled neurons per hemisphere, two of which are the bright PIL neurons (Fig. 5D). We find no detectable GMR19B09>myr::tdTomato expression in MIL neurons and only a very faint trace, if any, in the posterior distal VNC pair (Supplementary Fig. 12). Of the ∼200 GMR19B09-positive VNC cells, roughly 60 co-express sfGFP::Lgr3 at low, yet detectable levels. Any of the ∼70 CNS neurons coexpressing sfGFP::Lgr3 and GMR19B09> are good candidate cells to convey the Dilp8 signal.
Since GMR19B09 clearly colocalizes with PIL neurons, one of the cell populations that most strongly expresses Lgr3, we looked for other Gal4 lines that could allow the genetic manipulation of these neurons. We noticed that the MZ-699-Gal4 (MZ699>) line drives expression in similar type of neurons, named #5 neurons22. Like PIL neurons, #5 neurons contribute to projections at the anteriormost region of the brain neuropil and to the median bundle tract that follows the esophageal foramen22. Immunofluorescence analyses of dissected CNS of larvae carrying MZ699>myr::tdTomato and sfGFP::Lgr3 transgenes demonstrate that PIL neurons are a subset of #5 neurons (Fig. 5E). To test whether silencing Lgr3 in MZ699> cells could rescue the tissue-damage-dependent developmental delay, we drove Lgr3-IR-V22 under the control of MZ699> and exposed the larvae to EMS. We find that MZ699>Lgr3-IR-V22 strongly suppresses the EMS-dependent delay (Fig. 5F). Apart from #5 neurons, colocalization of both transgenes in the brain region was only detected in 3 other neurons that appear to be a subset of #2 neurons, defined as medial antennocommissural tract relay interneurons whose fibers arborize in the laternal horn region22. However, GMR19B09> does not appear to label these cells, making it unlikely that they contribute to the Lgr3-dependent Dilp8 signal transduction. Other subesophageal neurons and many other VNC neurons, with the notable exception of the strong distal VNC neuronal pair, express both MZ699>myr::tdTomato and sfGFP::Lgr3. As GMR19B09> labeling of the subesophageal region is sparce21, and neither MZ699> or GMR19B09> drives detectable expression in MIL neurons (Supplementary Fig. 12 and Fig. 13), this suggests that either the PIL neuron expression overlap could be functionally relevant or that the Dilp8 signal requires Lgr3 expression in any of the ∼70 sfGFP::Lgr3 positive VNC cells.
Discussion
Different organs need to sense growth perturbations in distant tissues to coordinate their size and differentiation status during development1. Here, we have determined that the sensing of peripheral growth perturbations requires a novel population of CNS neurons expressing the Lgr3 relaxin receptor. Neuronal Lgr3 is required for the transmission of the peripheral growth aberration signal, Dilp8, to the prothoracic gland, which controls the onset of metamorphosis and thereby the cessation of imaginal disc growth1,3,4,9,23 (Fig. 5G). This work reveals a new Dilp8-Lgr3 pathway that is critical to ensure developmental stability in Drosophila. Our study opens many questions for further research, such as the determination of whether or not the interaction between Lgr3 and Dilp8 is direct, which of the ∼70 Lgr3-positive neurons are activated during Dilp8 expression, and how these connect to the ring gland. In this sense, the neuroanatomy of the sfGFP::Lgr3+ cells which also express GMR19B09> poses a problem of accessibility: if the activation of Lgr3 neurons by Dilp8 is direct, Dilp8 would have to somehow make its way across the blood-brain-barrier to activate these neurons deep in the brain. Alternatively, the Dilp8 signal could be received by other cells (either glial cells or other neurons with projections exposed to the hemolymph), and relayed through one or more steps before reaching the Lgr3+ cells (Fig. 5G, “Relay1?”). Furthermore, our study strongly suggests that the Lgr3+ cells that express both MZ699> and GMR19B09> are critical for the tissue damage developmental delay response. The fact that we were unable to detect GMR19B09>myr::tdTomato expression in the ring gland or in neurons innervating the ring gland strongly suggests that the elav-positive Lgr3+ neurons that are required for the Dilp8-dependent delay do not connect directly to the ring gland. Hence, it is likely that the Lgr3 neurons also need to relay the tissue stress signal at least once to the ring gland, either by secreting a second factor or connecting to a ring-gland innervating neuron (Fig. 5G, “Relay2?”). Together, these results indicate that the peripheral Dilp8 tissue damage signal is transduced through multiple steps before it reaches the ring gland, revealing unprecedented complexity and providing both important functional insight into the transduction of the Dilp8-dependent aberrant tissue growth signaling pathway, and opening fertile ground for further research.
The similarities between the neuroendocrine mechanisms controlling the larval to pupal transition in Drosophila and the hypothalamic-pituitary axis in vertebrates has been highlighted2,24. The neurosecretory cell-rich pars intercerebralis, in which the Lgr3-expressing PIL neurons are located, has anatomical, developmental and functional analogies to the hypothalamus, the structure that integrates the vertebrate CNS to the endocrine system via the pituitary gland. Similarly, the Drosophila pars intercerebralis connects the CNS to the endocrine ring gland complex via neurosecretory cells25, such as the IPCs16,25. Both systems have roles in stress-response, energy metabolism, growth, water retention, and reproduction5,24. The neuroanatomy of Lgr3-positive neurons, such as the PIL neurons (Fig. 3F-H), suggests they are well positioned to relay signals or to modulate the activity of ring gland-innervating neurons during tissue stress events that trigger Dilp8 secretion from the periphery. Candidate neurons that could interact with PIL neurons are the IPCs, PTTH neurons and DMA1 neurons, just to cite a few. Apart from arborizing in the pars intercerebralis region (Fig. 3F-H), PIL neurons send projections via the median bundle to the subesophageal region21,22. This region is known to harbor the serotonergic SE0-PG neurons, which directly innervate the PG, thereby regulating developmental timing as a response to nutritional cues17. It will be interesting to test whether PIL and SE0-PG neurons synapse in the subesophageal region and whether the latter also have a role in the tissue damage response.
As the timing of vertebrate developmental transitions, such as puberty, can also be altered by intrinsic and extrinsic factors affecting body growth, such as inflammatory disease and nutritional status2, the exploration of the role of relaxin signaling in modulating the hypothalamic-pituitary axis is a promising area for research. This is highlighted by the fact that the hypothalamus expresses relaxin receptors, including the Lgr3-homologue, RXFP1, in mammals and fish5,26, suggesting that a central neuroendocrine role for relaxin receptors might have evolved before the vertebrate and invertebrate split. A candidate peptide to regulate hypothalamic-pituitary stress-responses via relaxin receptors is the neuropeptide Relaxin-3 (RLN3), which has been traditionally viewed as being the ancestor ligand for all vertebrate relaxins27,28. RLN3 is strongly expressed in stress-responsive neurons from the nucleus incertus that directly innervate and modulate hypothalamic activity5,29-31. Our results therefore reveal an unexpected and striking similarity between the Dilp8-Lgr3 pathway and the vertebrate relaxin signaling pathway and hint to an ancient stress-responsive pathway coordinating animal growth and maturation timing.
Author contributions
AG, FH, APC, AM, CN, MG, AMD, YV, TK and AMG performed experiments, analyzed data and contributed intellectually to the paper. AG, FH, APC, CN, TK and AMG were involved in pupariation time assays. FH, APC, AM, MG and AMD were involved in the generation and molecular characterization of mutants and transgenes. AG, APC, AM, YV and AMG were involved in FAi measurements. FH, APC, and AMG were involved in imaging. AG and AMG wrote the paper with the help of all authors.
Methods
Stocks
The Drosophila melanogaster stocks w1118 Bx-gal4 (w1118 P{GawB}BxMS1096), UAS-rpr (w1118; P{UAS-rpr.C}14), Lgr3-IR-V10 (y1 v1;{TRiP.JF03217}attP2), Lgr3-IR-V22 (y1 sc* v1; {TRiP.GL01056}attP2/TM3 Sb1), elav-Gal4 (P{w[+mW.hs]=GawB}elavC155), elav-Gal4 (P{w[+mC]=GAL4-elav.L}2/CyO), w1118;Mi{ET1}lgr3MB06848, w1118; snaSco/SM6a, P{w[+mC]=hsILMiT}2.4, tub-Gal4 (y1 w*; P{tubP-GAL4}LL7/TM3, Sb[1]), w1118; Lgr3Df(3)BSC321/TM6C Sb1 cu1, Gad1-Gal4 (P{w[+mC]=Gad1-GAL4.3.098}2/CyO), Cha-Gal4 (w1118; P{w[+mC]=Cha-GAL4.7.4}19B/CyO P{ry[+t7.2]=sevRas1.V12}FK1), GMR19B09-Gal4 (w1118; P{y[+t7.7] w[+mC]=GMR19B09-GAL4}attP2), and GMR17G11-Gal4 (w[1118]; P{y[+t7.7] w[+mC]=GMR17G11-GAL4}attP2) were obtained from the Bloomington Drosophila Stock Center at Indiana University. The stock y1 M{w[+mC]=Act5C-Cas9.P}ZH-2A w* (reference #13) was obtained from Bestgene. The stock arm-gal4 (w*; P{w[+mW.hs]=GAL4-arm.S}11) was a gift from P. Domingos. The stocks UAS-dilp8b::3XFLAG and UAS-dilp8c::3XFLAG (reference #3) were a gift from M. Dominguez. The stock w*; P{GawB}Mz699 and UAS-myr::tdTomato/CyO; TM2/TM6B were a gift from M.L. Vasconcelos. Balanced lines were generated by crosses to the stock w1118; If/CyO; MKRS/TM6B, which was a gift from A. Jacinto. Stocks are maintained at low densities at 18°C in a 12-h light:dark cycle.
Generation and molecular characterization of the Lgr3ag1 allele
We generated a mutation in the Drosophila Lgr3 locus by using the MiET1 transposase to remobilize the MB Minos element Lgr3MB06848 (reference #8), which is inserted in the seventh Lgr3 intron, <100 bp from exon 7 (Fig. 1B and Supplementary Fig. 1A). Virgin Lgr3MB06848 females were crossed with males carrying the heat shock-inducible MiET1 transposase on a Cy balancer chromosome (w1118; If/Cy-Minos; MKRS/TM6B) and transferred to new vials every day8. After 48 h of development and until pupariation, the F1 progeny was given a 1-h 37°C heat-shock daily to induce the expression of the MiET1 transposase. Cy-Minos/+; Lgr3MB06848/MKRS or TM6B male adults were selected and individually crossed to the balancer strain w1118; If/CyO; MKRS/TM6B. A single eGFP-negative (lacking the Mi{ET1} element) male was selected from each cross and mated with w1118; If/CyO; MKRS/TM6B females. The putative Lgr3 excisions were balanced over TM6B to obtain the following genotypes w1118; +/+; Lgr3MB06848 excision/TM6B. We obtained one imprecise excision that generated the Lgr3ag1 mutant allele and three precise excisions named Lgr3ag2, Lgr3 ag3, and Lgr3ag4 all of which behaved the same way. In this study, the Lgr3ag2 line was used as the genetic background control for the imprecise excision allele Lgr3ag1.
To molecularly characterize the Lgr3ag1 deletion, we performed a series of polymerase chain reaction assays with primer pairs located around the Lgr3MB06848 insertion (Supplementary Fig. 1A). Lgr3ag1 was initially detected by the failure to amplify a 866-bp PCR product flanking the Lgr3MB06848 insertion (green arrows, Supplementary Fig. 1A), and then a 240-bp PCR product using a pair of primers on exon 8 and 9 to the right of the Lgr3MB06848 element (red arrows, Supplementary Fig. 1A, B), indicating that a deletion occurred downstream of the element following its remobilization. A PCR using a primer pair in the exon 11 produced a positive result, indicating that exon 11 was present in Lgr3ag1 (not shown). This suggested that while the Lgr3ag1 deletion was very large, its breakpoints were confined within the Lgr3 locus. We then tried to amplify a 5.3-kb PCR product with a primer upstream of the Lgr3MB06848 position (green forward arrow, Supplementary Fig. 1A-C) and another on exon 11 (purple arrow, Supplementary Fig. 1A-C). Instead of the predicted >5.3 kb product, we obtained a ∼1.6 kb product (Supplementary Fig. 1C), demonstrating that Lgr3ag1 is a large deletion of approximately 3.8 kb in the Lgr3 locus (Supplementary Fig. 1D). This alone indicates that at least 69 aminoacids (aa; I327-T395) are lacking in the Lgr3ag1 Leucine Rich Repeat (LRR) domains, which are critical for relaxin ligand binding in vertebrate relaxin receptors5.
Next, we performed reverse-transcriptase (RT)-PCR analyses with mRNA isolated from Lgr3ag1 and controls Lgr3+/+ and the original Lgr3MB06848 stock. Lgr3ag1 generated a smear with a major band that was ∼200 bp smaller than the other control genotypes (Supplementary Fig. 2A; Supplementary Fig. 2B, C are RT- and rp49 control reactions). Sanger sequencing determined that the Lgr3ag1 deletion leads to readthrough from exon 7 directly into intron 7 and usage of an aberrant splicing donor within intron 7 directly into exon 9 (Supplementary Fig. 2D). The resulting transcript therefore has an intron-encoded premature termination codon (PTC) that truncates the Lgr3 protein one amino-acid after D326 (Fig. 1E). We conclude that Lgr3ag1 encodes a severely truncated protein without the seven transmembrane (7TM) domains and G protein coupling Carboxy terminus (Fig. 1E).
Generation of pUASP-Lgr3 and pUASP-dilp8 flies
A Drosophila and Homo sapiens codon-optimized cDNA corresponding to full-length Lgr3 was synthetized de novo and cloned into pUASP32 using KpnI and NotI sites to make pUASP-Lgr3. This plasmid was injected into w1118 and two independent insertions were tested, pUASP-Lgr3a (M3, on Chr III) and pUASP-Lgr3b (M6, on Chr II). A similar protocol was used to place the dilp8::3xFLAG construct described in reference #3 into pUASP, making pUASP-dilp8a.
Generation of sfGFP::Lgr3 and PTC-inducing alleles
We used a CRISPR/Cas9 mediated homologous repair strategy12,13 to tag the endogenous Lgr3 protein at its Amino (N)-terminus with superfolder green fluorescent protein (sfGFP)14 followed by a flexible linker spacer sequence GSGSGS (Fig. 3A and Supplementary Fig. 5). The following guide RNAs (gRNA1 and gRNA2, designed with http://tools.flycrispr.molbio.wisc.edu/targetFinder/)12 were synthetized and cloned into pU6-BbsI-chiRNA:
gRNA 1
Fw CTTCGGAGCACTCAATTCCCACTC (CGG)
Rv AAACGAGTGGGAATTGAGTGCTCC
gRNA 2
Fw CTTCGCAAACTCAAGTAGAATATCA (CGG)
Rv AAACTGATATTCTACTTGAGTTTGC
The PAM regions (blue) are located right after the forward primers of both gRNAs 1 and 2, as shown above. Red nucleotides are used for cloning purposes.
As a repair cassette we designed the following sequence, where each color represents the following:
Yellow: Homology region up to Lgr3 ATG site
Red: Lgr3 ATG
Green: sfGFP
Blue: Spacer (GSGSGS)
Violet: Homology region after Lgr3 ATG site
CACTTAAAACTCTTCTCCGCGAGCTGTGAACATTAGCCAAATGAAGTGACAAGAAATTAACGCAAAAATAAAACAAGAAGACGGAGCGGTATAAGAAATAATAATATAAAAACTCAATGAGTCAGCACCGCATCAGCTCCTGCTGCTGTTGTTCTTCTTATTGCTGTTGTTTGTGGGGGCGTGGCCGGAGTGGGAATTGAGTGCTCCTAATGATGAACTCGGTCAAGGAGCCAGTGCAGCCATGGTGGCCAAGTAATTAGATAAGCGAGCGTGCAAAACAGGAGCAAACCGATAAATCGCCATGCGTAAGGGCGAGGAGTTGTTCACGGGAGTTGTGCCCATATTGGTTGAGCTGGATGGAGATGTGAATGGCCACAAGTTCAGTGTGCGGGGTGAGGGAGAAGGAGACGCAACAAACGGTAAGCTGACACTGAAGTTCATTTGTACTACGGGCAAGCTCCCGGTGCCATGGCCCACATTGGTCACCACCCTGACCTATGGCGTGCAATGCTTCGCCCGATATCCAGATCATATGAAGCAGCATGATTTCTTTAAGTCGGCCATGCCCGAGGGTTACGTACAAGAGCGCACTATTAGCTTTAAGGACGACGGTACGTATAAAACCAGGGCTGAGGTGAAGTTTGAGGGTGATACCCTGGTGAACCGCATTGAATTGAAGGGCATCGATTTTAAGGAGGACGGCAACATCCTGGGCCACAAGCTCGAATATAATTTTAATAGCCATAATGTTTACATTACCGCGGACAAGCAGAAGAATGGAATTAAGGCTAATTTCAAGATCCGACATAATGTGGAGGACGGATCCGTTCAGTTGGCCGATCACTACCAGCAAAACACCCCCATCGGAGATGGCCCCGTCCTGCTGCCCGATAACCACTACCTGAGTACCCAGTCCGTCCTGTCGAAGGATCCTAATGAGAAGCGGGATCATATGGTGCTGCTGGAGTTTGTGACTGCCGCCGGCATAACGCATGGAATGGACGAGCTGTATAAAGGCTCCGGTAGTGGTTCCGTCTACGGCAGGAGCATCGCCGTAGGCTTCTGTCTGATGACCGTCGTCCTTCTGCTGGCCGCCGTGATATTCTACTTGAGTTTGGGTGAGTCCTTAGAGTGATGTCCTTTCAAAATTCCATCATTCGCAAACCTAAATAATTTCTGAATCAAGAATGTTCAAAATCTTAGCAATTATTATACGCATAATTTGTGAAACTACTTAAAGTTCTTTTAAAACTTGAGCTGCTGTAAATTTCTATATATACTTTCGTATCCTTAAAGGGTTCCTTCGCTTGAAGCAAAAACCAAAATCAAATTCCAAACTGCAAA
The repair cassette was synthetized de novo into a pUC57 plasmid and co-injected with the two gRNAs into the stock y[1] M{w[+mC]=Act5C-Cas9.P}ZH-2A w[*], which strongly and ubiquitously expresses a human codon optimised cas9 (reference #12). The adults originating from this injection were separately crossed to a MKRS/TM6B stock. Males from each F1 cross were separately crossed again to MKRS/TM6B. When larvae appeared in the vials, we extracted gDNA from the F1 male progenitor and made PCR reactions using the primer pairs:
Lgr3_crispr_testF - CCAATAACTTTAAGCCGTCTGTG
SuperfolderGFP_R - CAGCACCATATGATCCCGCT
SuperfolderGFP_F - CCTATGGCGTGCAATGCTTC
Lgr3_crispr_testR - TATAGCTGTGCGAATTTCTCGAT
These primers were designed to indicate the correct insertion of the sfGFP repair cassette from both sides in the genome. Positive hits were sequenced (ag5-ag9). Ten negative hits were also sequenced and two were retained as background controls (ag10 and ag11). The y1 M{w[+mC]=Act5C-Cas9.P}ZH-2A w* cassette was removed by selecting against eye color and stocks were kept as homozygous stocks, except for allele ag8, which was kept balanced over TM6B.
Measurement of the developmental timing of pupariation
Male and virgin female flies aged 3-10 days old were crossed and 1-2 days later transferred to an agar plate with yeast-sucrose paste (1:1). The next morning, the flies were transferred to a fresh plate to lay eggs for 3-6 h, depending on the experiment. To control for overcrowding, larvae were transferred to vials containing normal Drosophila food in 3-12 batches typically between 10-30 depending on the experiment. Survey of pupae consists in counting the number of pupae in each time interval. The final total n of pupae for each genotype in each experiment is depicted in the respective figures. For pupariation time assays, we aimed at obtaining at least 30 individual larvae per genotype, yet some genotypes were sick or difficult to obtain due to balancer chromosomes, yielding less larvae of the correct genotype to score. Male and females were scored together. The time of pupariation of each larvae was determined and genotypes were compared with the Kruskal-Wallis non-parametric test followed by post-hoc ranks test after Conover, with α = 0.01 or 0.05, as indicated in each figure, using the software Infostat. Data was plotted in box-plots representing median, 25 and 75% quartiles, and 5-95% percentiles as whiskers. Data points falling outside of the 5-95% interval were plotted as outliers. The Kruskal-Wallis test is a rank-based nonparametric test that can be used to determine statistically significant differences between two or more groups. It is a nonparametric alternative to the one-way ANOVA, and as such, normal distribution is not required. Developmental timing assays were performed at 25°C under constant light, except for experiments reported in Fig. 2A, and Supplementary Figure 3B, which were done in a 12-h light:dark cycle, and EMS experiments (see below), which were done in the dark to minimize the reaction of EMS with light.
Duration of the third instar
Egg collections were performed on normal food plates and larvae were reared at controlled densities without additional yeast (about 200 eggs/60 mm diameter normal fly medium plate). Newly molted third instar larvae were collected every 2 h as described previously33, 34. After staging, collected larvae were raised in a normal cornmeal/molasses medium at 20-30 larvae/vial without additional yeast. Pupariation time was observed every 2 h until all treated larvae pupariated or died. Male and females were scored together. We defined pupariation as cessation of movement with evaginated spiracles. These experiments were performed at 25°C under constant light.
Ethyl methanesulfonate (EMS) treatment
Larvae were collected as described above and transferred 72 h after egg laying to fresh food with 10 mM of EMS (Sigma) or PBS as control. Developmental time was measured as indicated above. EMS food was prepared as follows: food was melted and cooled to 55°C, an appropriate volume of freshly made EMS stock solution in PBS was added and thoroughly mixed and 3 ml per tube were dispensed. EMS and PBS tubes were kept in the dark as much as possible throughout the experiments.
Immunofluorescence staining
Brains of wandering 3rd instar larvae or 1st instar larvae were dissected in cold PBS, fixed for 30 min in 4% paraformaldehyde, rinsed with PBS with Triton (0.3%) (PBST), incubated with primary antibody overnight and with fluorescently labelled secondary antibody for 2 h. Nuclei were counterstained with DAPI (Sigma) and tissues mounted in Fluoromount-G (Southern Biotech). Antibodies used were: mouse anti-GFP 1:200 (DSHB, 12E6), mouse anti-nc82 1:250 (DSHB), rabbit anti-GFP 1:200 (Life technologies, A11122), rabbit anti-Dilp7 1:5000 (I. Miguel-Aliaga18), rat anti-Dilp5 1:400 (gift from P. Leopold35). Images were obtained with a Zeiss LSM 710 Confocal Microscope and images were analyzed using FIJI software36. Typically 5-10 brains were mounted for observation and one representative image per genotype is depicted in figures. Brains from male and female larvae were scored together.
Adult wing measurements
Pairs of the left and right wings of male individuals reared at 29°C were rinsed with ethanol and mounted in a glycerol: ethanol solution. Photos were obtained in a Zeiss Axiovert 40 CFL microscope. The wing areas and wing lengths were calculated as previously described3, 39, using Fiji. We used the Fluctuating Asymmetry index (FAi) to assess intra individual size variations between the left and right wings39. Namely, FAi = Var(Ai), where Ai are the normalized differences between left and right wing areas of each individual Ai=Aleft-Aright/[(Aleft+Aright)/2]. Results were compared statistically using the F-test for the significance of the difference between the FAi of the samples, an appropriate test for dispersion3, 39. Bonferroni corrections (α/n) for multiple comparisons was applied to Fig. 4D and Fig. 4F, using α = 0.05
To control for measurement errors, we measured the area of the same wing three times. Values obtained were 18310.2 ± 30.5 AU, which gives a coefficient of variation (CV) of 0.17%, which is smaller than the CV of wing areas of control Lgr3+/+ by a factor of ∼23 (CV = 3.88%, n = 48, flies reared at 29°C).
gDNA, cDNA, RT-PCR and qRT-PCR analyses
gDNA was extracted from a group of flies or single flies38. Briefly, the flies were macerated using pellet pestles and homogenized in 100 μl DNA Extraction Buffer (1 M Tris-HCl at pH 8.2, 0.5 M EDTA, 5 M NaCl). Then, we added 1 μl Protease K 50 ng/μl (Roche), and incubated the mixture at 37°C for 1 h, followed by 95°C for 5 min, to inactivate the protease.
RNA was extracted using the Direct-zol(tm) RNA MiniPrep kit (Zymo Research), following manufacturer’s instructions. The material used for the RT-PCR experiments described in Supplementary Fig. 2 was 15 virgin males aged between 3-7 days were macerated using pellet pestles and homogenized in 500 μl TRI Reagent® and centrifuged at 12000 g for 1 min, to lower tissue debris. An extra DNAse treatment (Turbo DNA-free kit, Ambion, Life Technologies) was performed to reduce gDNA contamination. cDNA synthesis was performed using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (ThermoScientific), following manufacturer’s instructions. For this study, PCR and RT-PCR primers were designed and their specificity tested using Primer - BLAST or primer3. A T100 Thermal Cycler(tm) (BioRad) for performing the PCR steps. The following primers were used for PCR analyses described in Supplementary Fig. 1-2.
Lgr3_salto_fw (expected product size 866 bp)
Fw CCGACGCCTTGCTGCTAACT
Rv TTTATGGAGCGGGCGTGGTC
Lgr3_exonshort Lgr3 (expected product size 331 bp)
Fw CCGACGCCTTGCTGCTAACT
Rv GTGCGTTATGAGGTTGTGCTG
Lgr3_exon3p Lgr3 (expected product size 240 bp)
Fw CGCCTTGTCGGTAATCCCAT
Rv GTGGCTCCATTAAACTGCTGC
Lgr3_exons (expected product size 5307)
Fw CCGACGCCTTGCTGCTAACT
Rv CAAAGACCACCAACCAGGCGTA
rp49 (control)
Fw TTGAGAACGCAGGCGACCGT
Rv CGTCTCCTCCAAGAAGCGCAAG
qRT-PCR experiments were performed using Lightcycler® 96 (Roche) using the FastStart Essencial DNA Green Master dye and polymerase (Roche). The final volume for each reaction was 10 μl, consisting of 5μl of dye and polymerase (master mix), 2 μl of cDNA sample, and 3 μl of the specific primer pairs. The following primers were used:
rp49 (control)
Fw TTGAGAACGCAGGCGACCGT
Rv CGTCTCCTCCAAGAAGCGCAAG
Lgr3
Fw GCTGGGTGCCCATCATCGTTAT
Rv CAAAGACCACCAACCAGGCGTA
Primer efficiency for qRT-PCR was tested by serial dilution. Data was expressed as %rp49 according to the formula: %rp49 = (2^-(ΔCqLgr3-ΔCqrp49))*100. The geometric mean ± the standard error of the mean of three biological repeats was used and data was analyzed by one-tailed unpaired Student’s t-test using Bonferroni corrections (α/n) for multiple comparisons, using α = 0.05. This test assumes the independent samples have equal variances.
General study design and statistics
In all experiments reported in this work, no data point was excluded. All data points, including outliers, are represented in the Figures and were used in the statistical analyses. No blinding was done and no particular randomization method was used to individuals to experimental groups.
Acknowledgments
We thank C. Mirth, C. Ribeiro and A. Jacinto for their comments and suggestions; I. Miguel-Aliaga, A. Jacinto, P. Leopold, P. Domingos, C. Mirth, R. Teodoro, M. Dominguez, and M.L. Vasconcelos, for reagents. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. AMG, FH, AM, AMD and TK are supported by the FCT, under the FCT Investigator Programme and FCT fellowships SFRH/BPD/94112/2013, PD/BD/52421/2013, SFRH/BD/94931/2013 and SFRH/BPD/74313/2010, respectively. AG is supported by the CONICET and UNS and YAV holds a CONICET fellowship. The work in the laboratory of AMG is funded by the CEDOC and the European Commission FP7 (PCIG13-GA-2013-618847). AG thanks NP Rotstein and LE Politi for providing funds and space to develop part of this project in their lab. The authors declare no competing financial interests.