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Leucine-rich repeat-containing G protein-coupled receptor 3: Biological Overview | References

BIOLOGICAL OVERVIEW

Body size constancy and symmetry are signs of developmental stability. Yet, it is unclear exactly how developing animals buffer size variation. Insulin-like peptide 8 (Dilp8) is responsive to growth perturbations and controls homeostatic mechanisms that co-ordinately adjust growth and maturation to maintain size within the normal range. This study shows that Leucine-rich repeat-containing G protein-coupled receptor 3 (Lgr3) is a Dilp8 receptor. By functional and cAMP assays, a pair of Lgr3 neurons were found to mediate the homeostatic regulation. These neurons have extensive axonal arborizations, and genetic and GFP reconstitution across synaptic partners (GRASP) show these neurons connect with the insulin-producing cells and PTTH-producing neurons to attenuate growth and maturation. This previously unrecognized circuit suggests how growth and maturation rate are matched and co-regulated according to Dilp8 signals to stabilize organismal size (Vallejo, 2015).

The impressive consistency and fidelity in size of developing organisms reflects both the robustness of genetic programs and the developmental plasticity necessary to counteract the variations in size arising from genetic noise, erroneous morphogenesis, disease, or injury. To counterbalance growth abnormalities, systemic homeostatic mechanisms are implemented that delay the onset of the reproductive stage of adulthood until a correct size of the individual and its body parts has been reached. Indeed, most animals initiate a pubertal transition only once a critical size and body mass has been achieved and generally, in the absence of tissue damage or growth abnormalities. However, the mechanisms underlying such homeostatic regulation have yet to be fully defined (Vallejo, 2015).

Recently, the secreted peptide Dilp8, a member of the insulin/relaxin-like family has been identified as a factor mediating homeostatic control in Drosophila melanogaster. During the larval (growth) stage, the expression of dilp8 declines as maturation proceeds, whereas its expression is activated when growth is disturbed. Hence, fluctuating Dilp8 levels provides a reliable read-out of overall growth status (e.g., deficit) and of the time needed to complete growth and Dilp8 also orchestrates hormonal responses that stabilize body size. This includes inhibiting the production of the steroid hormone ecdysone by the prothoracic gland (PG) until the elements or organs affected are recomposed and also slowing down growth rates of undamaged tissues to ensure affected organs catch up with normal tissues in order to the adult flies reach a normal body size, maintain body proportions and symmetry. Accordingly, in the absence of dilp8, mutant flies are incapable of maintaining such strict control over their size, as reflected by the exaggerated variation in terms of overall proportionality and imperfect bilateral symmetry. However, the receptor that transduces Dilp8 signals and its site of action remained unknown (Vallejo, 2015).

Two models can be envisioned that would account for the establishment of such homeostatic regulation: a 'central' mechanism that dictates coordinated adjustments in both the duration and rate of growth, and an 'endocrine' mechanism that involves sensing and processing Dilp8 signals directly by hormone-producing cells. In Drosophila, several anatomically separate neural populations regulate growth and maturation time by impinging directly on the ring gland (comprising the PG and the juvenile hormone-producing corpus allatum, CA). Thus, the receptors that transduce the Dilp8 signals of growth status may act directly or communicate with neurons that produce the prothoracicotropic hormone (PTTH) and/or the neurons of the pars intercerebralis, including the insulin-producing cells (IPCs), that synthesize and release insulin-like peptides Dilp2, Dilp3 and Dilp5. Insect PTTH neurons, which are analogous to the gonadotropin-releasing hormone (GnRH) neurons in mammals, signal the commitment to sexual reproduction by stimulating the production of ecdysone in the PG in order to terminate growth. The IPCs in the pars intercerebralis, a functional equivalent of the mammalian hypothalamus, integrate nutritional signals and modulate tissue growth accordingly. Manipulation of IPCs by genetic ablation, starvation, or mutations in the single insulin receptor leads to the generation of animals with smaller size. Similarly manipulations of the PTTH neuropeptide and neurons result in variations in size of the adult flies, leading to larger or smaller than normal flies due to an extension or acceleration of the larval period and delayed pupariation. The insulin receptor also directly activates synthesis of the juvenile hormone (JH) in the CA, a hormone that promotes growth and the juvenile programs, and of ecdysone production in the PG, again augmenting the variation in normal adult size. These observations may explain how environmental and internal influences by operating through individual IPCs or PTTH neurons enable body size variation and plasticity in developmental timing that can be vital for survival in changing environments. However, the origin of developmental stability and invariant body size may require different or more complex neural mechanisms from those involved in adaptive size regulation (Vallejo, 2015).

By employing a candidate approach and biochemical assays, this study demonstrates that the orphan relaxin receptor Lgr3 acts as a Dilp8 receptor. This study identifies the neuronal population molecularly defined by the lgr3 enhancer fragment R19B09 (Jenett, 2012) and shows it is necessary and sufficient to mediate such homeostatic regulation. Using a cyclic AMP sensor as an indicator of Lgr3 receptor activation in vivo and tools for circuit mapping, it was determined that a pair of these Lgr3 neurons is highly sensitive to Dilp8. These neurons display extensive axonal arborizations and appear to connect with IPCs and PTTH neurons to form a brain circuit for homeostatic body size regulation. These data identify the insulin genes, dilp3 and dilp5, the JH, and ecdysone hormone as central in developmental size stability. Collectively, these findings unveil a homeostatic circuit that forms a framework for studying how the brain stabilize body size without constraining the adaptability of the system to reset body size in response to changing needs (Vallejo, 2015).

The data presented provide strong evidence that Dilp8 signals for organismal and organ homeostatic regulation of size are transduced via the orphan relaxin receptor Lgr3 and that activation of Lgr3 in molecularly defined neurons mediates the necessary hormonal adjustments for such homeostasis. Human insulin/relaxin-like peptides are transduced through four GPCRs, RXFP1 to 4. RXFP1 and 2 are characterized by large extracellular domains containing leucine-rich repeats similar to fly Lgr3 and Lgr4 receptors, and like Lgr3 (this study), their activation by their cognate ligand binding results in an increase in cAMP production. RXFP3 is distinctly different in structure from fly Lgr3 and its biochemical properties are also distinct, but RXPF3 is analogous to fly Lgr3 in the sense that it is found in highest abundance in the brain, suggesting important central functions for relaxin 3/RXFP3. However, a function in pubertal development and/or growth control for vertebrate relaxin receptors is presently unknown (Vallejo, 2015).

The neuronal populations that regulate body size and, in particularly, how their regulation generate variations in body size (plasticity) in response to internal and environmental cues such as nutrition have been intensely investigated. Less is known about how the brain stabilizes body size to ensure developing organisms reach the correct, genetically determined size. In particular, it remains unknown how limbs, and other bilaterally symmetric traits, grow to match precisely the size of the contralateral limb and maintain proportion with other parts even when they are faced with perturbations. Paired organs are controlled by an identical genetic program and grow in the same hormonal environment, and yet, small deviations in size can happen as result of developmental stress, genetic noise, or injury. Imperfections in symmetry thus reflect the inability of an individual to counterbalance variations and growth abnormalities (Vallejo, 2015).

This study shows that without lgr3, the brain is unable to detect growth disturbances and more importantly, it is not able to adjust the internal hormonal environment to allocate additional time during development to restore affected parts or catch-up on growth. Without lgr3, the brain also cannot slow down the growth rate to compensate for the extra time for growth so that unaffected and affected tissues can grow in a harmonious manner so as to sustain normal size, proportionality and symmetry. Using a cAMP sensor, this study has been able to define a pair of neurons that are highly sensitive to Dilp8 (Vallejo, 2015).

Communication in neuronal networks is essential to synchronize and perform efficiently. Notably, although most neurons have only one axon, Lgr3 responding neurons display extensive axonal arborizations reminiscent of hub neurons (Bonifazi, 2009). GRASP analyses show that Lgr3 neurons are broadly connected with the IPCs, and to a lesser extent with PTTH neurons, linking (Dilp8) inputs to the neuronal populations that regulate the key hormonal outputs that modulate larval and imaginal disc growth. Furthermore, the information flow from Lgr3 neurons to IPCs and to PTTH may explain how the brain matches growth with maturation in response to Dilp8. This brain circuit provides the basis for studying how the brain copes with genetic and environmental perturbations to stabilize body size, proportions and symmetry that is vital for the animal's survival (Vallejo, 2015).

Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing

How different organs in the body sense growth perturbations in distant tissues to coordinate their size during development is poorly understood. This study mutated an invertebrate orphan relaxin receptor gene, the Drosophila Leucine-rich repeat-containing G protein-coupled receptor 3 (Lgr3) and found body asymmetries similar to those found in insulin-like peptide 8 (dilp8) mutants, which fail to coordinate growth with developmental timing. Indeed, mutation or RNA intereference (RNAi) against Lgr3 suppresses the delay in pupariation induced by imaginal disc growth perturbation or ectopic Dilp8 expression. By tagging endogenous Lgr3 and performing cell type-specific RNAi, this Lgr3 activity was mapped to a new subset of CNS neurons, four of which are a pair of bilateral pars intercerebralis Lgr3-positive (PIL) neurons that respond specifically to ectopic Dilp8 by increasing cAMP-dependent signalling. This work sheds new light on the function and evolution of relaxin receptors and reveals a novel neuroendocrine circuit responsive to growth aberrations (Garelli, 2015).

Different organs need to sense growth perturbations in distant tissues to coordinate their size and differentiation status during development. This study has 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 growth. This work reveals a new Dilp8-Lgr3 pathway that is critical to ensure developmental stability in Drosophila. This study opens many questions for further research, such as the determination of which of the eight bilateral Lgr3-positive interneuron populations are critical during Dilp8 expression, whether or not the interaction between Lgr3 and Dilp8 is direct and how Lgr3-positive neurons relay information to the ring gland (Garelli, 2015).

Of the eight bilateral Lgr3-positive interneuron populations identified in in this study, the cholinergic PIL neurons both require Lgr3 for the Dilp8-dependent developmental delay activity and respond to Dilp8 by increasing cAMP levels. Therefore, PIL neurons are the best candidates to mediate the Dilp8-dependent developmental delay. Further research is necessary to determine if PIL neurons are sufficient to regulate developmental timing in the absence of growth aberrations or ectopic Dilp8 signals (Garelli, 2015).

While the results clearly indicate that Dilp8 and Lgr3 act on the same pathway, their biochemical relationship is less clear. As Dilp8 is an Ilp and Lgr3 is a homologue of a vertebrate receptor for an Ilp (relaxin), it is tempting to propose a direct ligand-receptor interaction between them. This possibility is supported by the strong genetic interaction between dilp8 and Lgr3 and the finding of Dilp8-responsive Lgr3-positive neurons. However, this study also raises at least three possible issues with this interpretation of the data. First, the neuroanatomy of the CNS neuronal populations requiring Lgr3 activity suggests that Dilp8 could have to traverse the blood-brain barrier to activate Lgr3-positive interneurons deep in the brain, something which is presently unclear if it can be achieved. Alternatively, the data can also be explained if the Dilp8 signal is received by other cells (if by the CNS, these can be either glial cells or other neurons with projections exposed to the haemolymph), and relayed through one or more steps before reaching the Lgr3-positive cells. A similar route through blood-brain barrier glial cells has been proposed to explain the relay into the CNS of a fat-body-derived signal that controls neuroblast reactivation (Garelli, 2015).

Second, Lgr3 was not identified among candidate Dilp8-binding cell surface receptors/co-receptors. Clearly, the biochemical identification of alternative cell surface-binding proteins such as the InR, Nrg and the RYK-like Drl does not rule out the possibility of a direct interaction between Dilp8 and Lgr3 in vivo, nevertheless it strongly indicates that Dilp8 can consistently interact with a likewise strong receptor candidate for an Ilp, such as the InR. The LRC technique that was used can identify receptors of interest with affinities spanning 4 orders of magnitude at expression levels as low as 2,000 receptors per cell (www.dualsystems.com). However, it is not yet clear how quantitative it can be relative to affinity constants. Dilp8 has been previously shown to modulate growth in vivo often in opposite ways depending on the observed tissue. Namely, Dilp8 ectopic expression throughout development leads to heavier adults and to reduced expression of the translational inhibitor and FOXO-target Thor (4E-BP) in the larval fat body, which is consistent with a local increase in insulin/IGF-like signalling. In contrast, Thor levels are higher in imaginal discs in the same animal. These results show that understanding the relationship between Dilp8, InR and Lgr3 will be a challenge for further studies. One possibility, if Dilp8 can indeed interact with Lgr3 in other contexts, is that there is a crosstalk between Lgr3 and InR receptors. The other possibility is that Dilp8 has a low-affinity interaction with the InR, which could be potentiated in certain physiological conditions. Affinity profiling of the Dilp8 and InR interaction, as well as that of Lgr3, should bring insight into this scenario. As regards the other Dilp8-specific candidate receptor, Drl, it also binds to Wnt5 to control aspects of axonal guidance, raising the possibility that the interaction between Dilp8 and Drl, if confirmed, can interfere with circuit formation. Interestingly, Drl has been shown to be expressed in four large glial cells in the interhemispheric region of the brain, close to the PIL neurons, and to be dynamically regulated between the third instar larvae and early pupae. Therefore, the interaction between Dilp8 and Drl should be carefully followed-up and independently verified (Garelli, 2015).

Third, the fact that ectopic expression of Dilp8 only leads to a detectable increase in cAMP signalling in PIL neurons, and not in other Lgr3-positive neurons, indicates that Lgr3 activation by Dilp8 requires other molecular and/or cellular players. Any of the factors identified biochemically in this study could participate in PIL neuron selectivity, for instance, as a differentially enriched co-receptor. Alternatively, PIL neurons could be selectively activated downstream of other cellular players, via a mechanism which could involve a signal relay by direct synapsis or proximity to other cells that participate in the transduction of the Dilp8 signal from the periphery to the ring gland. In this case, Dilp8 would probably activate Lgr3-positive neurons indirectly. Therefore, in the absence of further evidence suggesting a direct relationship between Dilp8 and Lgr3, the possibility cannot be ruled out that Lgr3-positive neurons are not a direct target of Dilp8, but rather intermediary players in the Dilp8 developmental stability pathway (Garelli, 2015).

How the Dilp8 signal reaches the ring gland after having triggered activity in some of the eight bilateral Lgr3-positive neuronal groups remains to be determined. The fact that sfGFP::Lgr3 or GMR19B09>myr::tdTomato expression were not detected in the ring gland or in neurons innervating the ring gland, strongly suggests that the Lgr3-positive 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. 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 signalling pathway and opening fertile ground for further research (Garelli, 2015).

The similarities between the neuroendocrine mechanisms controlling the larval-to-pupal transition in Drosophila and the hypothalamic-pituitary axis in vertebrates has been highlighted. The neurosecretory cell-rich pars intercerebralis, in which the Dilp8-responding and 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 cells. Both systems have roles in stress response, energy metabolism, growth, water retention and reproduction. The neuroanatomy of Lgr3-positive neurons, such as the PIL neurons, 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. Apart from arborizing in the pars intercerebralis region, PIL neurons send projections via the median bundle to the subesophageal region. This region is known to harbour the serotonergic SE0-PG neurons, which directly innervate the PG, thereby regulating developmental timing as a response to nutritional cues. 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 (Garelli, 2015).

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 status, the exploration of the role of relaxin signalling 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 fish, 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 relaxins. RLN3 is strongly expressed in stress-responsive neurons from the nucleus incertus that directly innervate and modulate hypothalamic activity. The current results therefore reveal an unexpected and striking similarity between the Dilp8-Lgr3 pathway and the vertebrate relaxin signalling pathway and hint to an ancient stress-responsive pathway coordinating animal growth and maturation timing (Garelli, 2015).

Drosophila Lgr3 couples organ growth with maturation and ensures developmental stability

Early transplantation and grafting experiments suggest that body organs follow autonomous growth programs, therefore pointing to a need for coordination mechanisms to produce fit individuals with proper proportions. Drosophila insulin-like peptide 8 (Dilp8) has been identified as a relaxin and insulin-like molecule secreted from growing tissues that plays a central role in coordinating growth between organs and coupling organ growth with animal maturation. Deciphering the function of Dilp8 in growth coordination relies on the identification of the receptor and tissues relaying Dilp8 signaling. This study shows that the orphan receptor leucine-rich repeat-containing G protein-coupled receptor 3 (Lgr3), a member of the highly conserved family of relaxin family peptide receptors (RXFPs), mediates the checkpoint function of Dilp8 for entry into maturation.Two Lgr3-positive neurons were identified in each brain lobe that are required to induce a developmental delay upon overexpression of Dilp8. These neurons are located in the pars intercerebralis, an important neuroendocrine area in the brain, and make physical contacts with the PTTH neurons that ultimately control the production and release of the molting steroid ecdysone. Reducing Lgr3 levels in these neurons results in adult flies exhibiting increased fluctuating bilateral asymmetry, therefore recapitulating the phenotype of dilp8 mutants. This work reveals a novel Dilp8/Lgr3 neuronal circuitry involved in a feedback mechanism that ensures coordination between organ growth and developmental transitions and prevents developmental variability (Colombani, 2015).

This study identified two growth coordinating Lgr3 (GCL) neurons in the pars intercerebralis that make physical contacts with PTTH neurons and therefore serve as an intermediate relay between growing tissues and the PG. It will be of future interest to resolve how Lgr3 signaling in the GCL neurons mechanistically couples with PTTH activity and ecdysone production (Colombani, 2015).

Knocking down Lgr3 in the GCL neurons recapitulates the elevated levels of fluctuating asymmetry (FA) observed in dilp8 or lgr3 mutant animals. Moreover, ectopic Dilp8 expression also induces FA, which is buffered by reducing neuronal Lgr3 levels. This indicates that levels of Dilp8 signaling during development are finely tuned in order to restrict developmental noise (Colombani, 2015).

The results presented in this study are consistent with a model whereby Dilp8 signaling in GCL neurons acts on ecdysone production by modulating the activity of PTTH neurons. This explains the observed effect of Dilp8 on the timing of the larva-to-pupa transition. Interestingly, the data also suggest that Dilp8/Lgr3 signaling in the GCL neurons controls growth coordination of peripheral organs through systemic ecdysone levels. It was previously proposed that inter-disc growth coordination relies on systemic effects mediated by ecdysone, and several reports indicate that ecdysone is required for imaginal tissue growth both in flies and in Lepidoptera. Future research will aim at deciphering the exact mechanism by which systemic ecdysone controls local tissue growth in order to mediate organ growth coordination (Colombani, 2015).

Interestingly, the Dilp8-Lgr3-PG circuitry shares some characteristics with the hypothalamic-pituitary axis in humans in which the hypothalamus integrates and relays information about the state of the body to the pituitary gland through synaptic communication with other neurons or the release of hormones. The observation that mammalian RFPs are expressed in the hypothalamus opens the possibility that similar relaxin/RXFP-dependent mechanisms monitoring the growth status of peripheral tissues are at play in humans. In line with this, reduced body mass, stress induced by heavy surgery, inflammatory diseases, and dietary restrictions have all been associated with a delay in puberty. It is therefore proposed that the Dilp8-Lgr3 axis represents an ancient surveillance mechanism ensuring developmental stability that may be conserved in higher animals (Colombani, 2015).

Search PubMed for articles about Drosophila Lgr3

Bonifazi, P., Goldin, M., Picardo, M. A., Jorquera, I., Cattani, A., Bianconi, G., Represa, A., Ben-Ari, Y. and Cossart, R. (2009). GABAergic hub neurons orchestrate synchrony in developing hippocampal networks. Science 326: 1419-1424. PubMed ID: 19965761

Colombani, J., Andersen, D. S., Boulan, L., Boone, E., Romero, N., Virolle, V., Texada, M. and Leopold, P. (2015). Drosophila Lgr3 couples organ growth with maturation and ensures developmental stability. Curr Biol 25: 2723-2729. PubMed ID: 26441350

Garelli, A., Heredia, F., Casimiro, A. P., Macedo, A., Nunes, C., Garcez, M., Dias, A. R., Volonte, Y. A., Uhlmann, T., Caparros, E., Koyama, T. and Gontijo, A. M. (2015). Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing. Nat Commun 6: 8732. PubMed ID: 26510564

Jenett, A., et al. (2012). A GAL4-driver line resource for Drosophila neurobiology. Cell Rep 2: 991-1001. PubMed ID: 23063364

Vallejo, D. M., Juarez-Carreño, S., Bolivar, J., Morante, J. and Dominguez, M. (2015). A brain circuit that synchronizes growth and maturation revealed through Dilp8 binding to Lgr3. Science [Epub ahead of print]. PubMed ID: 26429885

date revised: 18 January, 2016

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