InteractiveFly: GeneBrief

rickets: Biological Overview | References


Gene name - rickets

Synonyms - DLGR

Cytological map position - 34E5-34E6

Function - G-protein coupled receptor

Keywords - CNS, muscle wing expansion, GPCR, cuticle hardening, melanization

Symbol - rk

FlyBase ID: FBgn0003255

Genetic map position - chr2L:13,982,726-14,001,252

Classification - 7 transmembrane receptor (rhodopsin family)

Cellular location - surface transmembrane



NCBI links: Precomputed BLAST | EntrezGene

Recent literature
Anllo, L. and Schupbach, T. (2016). Signaling through the G-protein-coupled receptor Rickets is important for polarity, detachment, and migration of the border cells in Drosophila. Dev Biol [Epub ahead of print]. PubMed ID: 27130192
Summary:

Cell migration plays crucial roles during development. An excellent model to study coordinated cell movements is provided by the migration of border cell clusters within a developing Drosophila egg chamber. In a mutagenesis screen, two alleles were isolated of the gene rickets (rk) encoding a G-protein-coupled receptor. The rk alleles result in border cell migration defects in a significant fraction of egg chambers. In rk mutants, border cells are properly specified and express the marker Slbo. Yet, analysis of both fixed as well as live samples revealed that some single border cells lag behind the main border cell cluster during migration, or, in other cases, the entire border cell cluster can remain tethered to the anterior epithelium as it migrates. These defects are observed significantly more often in mosaic border cell clusters, than in full mutant clusters. Reduction of the Rk ligand, Bursicon, in the border cell cluster also resulted in migration defects, strongly suggesting that Rk signaling is utilized for communication within the border cell cluster itself. The mutant border cell clusters show defects in localization of the adhesion protein E-cadherin, and apical polarity proteins during migration. E-cadherin mislocalization occurs in mosaic clusters, but not in full mutant clusters, correlating well with the rk border cell migration phenotype. This work has identified a receptor with a previously unknown role in border cell migration that appears to regulate detachment and polarity of the border cell cluster coordinating processes within the cells of the cluster themselves.


BIOLOGICAL OVERVIEW

Bursicon is a hormone that modulates wing expansion, cuticle hardening, and melanization in Drosophila melanogaster. Bursicon activity is mediated through its cognate G protein-coupled receptor, Rickets. This study developed a membrane tethered Bursicon construct that enables spatial modulation of Rickets mediated physiology in transgenic flies. Ubiquitous expression of tethered Bursicon throughout development results in arrest at the pupal stage. The few organisms that eclose fail to undergo wing expansion. These phenotypes suggest that expression of tethered Bursicon inhibits Rickets mediated function. Consistent with this hypothesis, this study showed in vitro that sustained stimulation of Rickets by tethered Bursicon leads to receptor desensitization. Furthermore, tissue specific expression of the tethered Bursicon inhibitor unraveled a critical role for Rickets in a subset of adult muscles. Taken together, these finds highlight the utility of membrane tethered inhibitors as important genetic/pharmacological tools to dissect the tissue specific roles of GPCRs in vivo (Harwood, 2014).

Bursicon is a heterodimeric cystine-knot protein required for wing expansion and cuticle hardening in a variety of insects. Early studies performed in blowflies and cockroaches, showed a hormone of unknown molecular identity released from the central nervous system was important for tanning and wing expansion. More than four decades later the molecular identity of Bursicon and its cognate receptor, rickets were subsequently identified in Drosophila melanogaster. Rickets (Rk or dLGR2) is a member of the leucine rich repeat-containing subfamily of G protein coupled receptors (GPCRs) which is expressed both in the CNS and in the periphery (Diao, 2012). Previous studies have shown that a membrane anchored single subunit fusion construct of the Bursicon heterodimer (CFP-tBur-&betal-α) can activate Rk in vitro in a concentration dependent manner (Harwood, 2013). Membrane tethered ligands (MTLs) are cDNA constructs that express genetically encoded peptide hormones anchored to a transmembrane domain via a protein linker. The generation of this complex Bursicon heterodimeric tethered construct was done as part of an ongoing effort to sequentially develop a broad range of MTLs that selectively activate either insect or mammalian GPCRs (Harwood, 2014).

A major advantage of using MTL technology in vivo is that it enables activation of receptors in a targeted tissue without the confounding effects of soluble ligand diffusion. The transgenic Gal4/UAS system in conjunction with membrane tethered Bursicon offer an excellent model system in which to better understand the tissue dependence of Rk mediated signaling. In Drosophila, wing expansion occurs within 1 hour following eclosion. A tightly choreographed motor program is required for wing expansion and cuticle hardening. This series of events coincides with a biphasic release of Bursicon from a subset of crustacean cardioactive peptide (CCAP) positive neurosecretory cells. Bursicon is first released from the subesphogeal ganglion, followed by secretion from the abdominal ganglion into the hemolymph. The Drosophila circulatory system then disperses Bursicon throughout the organism (Peabody, 2008; Peabody, 2009). While most studies have focused on the location of Bursicon release, much less research has addressed the importance of tissue selective Rk activation (Honegger, 2008; Harwood, 2014 and references therein).

Previously it was thought that Rk was only required following eclosion, as a trigger for wing expansion and cuticle hardening. This postulate was based on data from two fly stocks, rk1 and rk4 which were thought to be receptor nulls (Baker, 2002). However a more recent study has shown that these flies are hypomorphs. In fact global knockdown of rk in vivo using RNAi results in developmental arrest, rather than just impaired wing expansion, melanization, and cuticle hardening (Loveall, 2010). A recent study also showed that deletion of the Bursicon β subunit results in significant lethality throughout pupariation, specifically during ecdysis (Lahr, 2012; Loveall, 2010). There has been no comprehensive study which has specifically examined what tissues require rk for proper development. A previous study which utilized a GFP reporter, demonstrated rk expression in the epidermis (Diao, 2012). More detailed analysis of rk transcript levels as revealed by Fly Atlas and RNA-Seq studies have shown that this receptor is expressed at low levels throughout development. However, which tissues or cells require rk expression for proper development remains an unexplored area of inquiry (Harwood, 2014).

Use of the Bursicon MTL, CFP-tBur-&betal-α), offered a novel approach to investigate the tissue specific requirements of rk. The use of tissue specific Gal4 drivers to target expression of the fused heterodimer provided a means to selectively modulate rk without the confounding effects of soluble ligand diffusion. Parallel studies using rk RNAi transgenic flies enabled a complementary approach to confirm conclusions drawn through the use of the membrane tethered ligand (Harwood, 2014).

Prior investigations have shown that a membrane tethered agonist can trigger long term receptor activation in flies. In this current study, it is illustrated that there is an alternative potential consequence that may result from membrane tethered ligand expression in vivo. Specifically the Bursicon MTL triggers Rk desensitization resulting in functional blockade of the receptor. This mechanism is supported by in vitro data demonstrating that long term Bursicon stimulation, using either soluble ligand or the corresponding MTL, essentially eliminated further Bursicon mediated signaling. This study shows that ubiquitous receptor inactivation using the CFP-tBur-&betal-α) in vivo results in developmental lethality. Furthermore, by expressing membrane tethered Bursicon with a collection of increasingly focused tissue specific Gal4 drivers, it was possible to show an essential role during eclosion and subsequent wing expansion. Parallel studies with rk RNAi constructs support each of the above conclusions (Harwood, 2014).

This study utilized a membrane tethered Bursicon construct (CFP-tBur-&betal-α) o probe the role of rk in development. Previous studies have shown that when assessed in vitro; CFP-tBur-&betal-α is an rk agonist (Harwood, 2013). Initial in vivo investigations revealed that expression of CFP-tBur-&betal-α in otherwise rk/Bursicon wildtype Drosophila (W1118) led to lethality and wing expansion defects, both unanticipated phenotypes. Although it is difficult to predict the effect of hormonal imbalance in vivo, it was hypothesized that the agonist activity of CFP-tBur-&betal-α would provide a gain of function and would be useful to rescue Bursicon mutant fly lines. Contrary to this expectation, the phenotypes resulting from ubiquitous expression of CFP-tBur-&betal-α were loss of function (i.e. lethality and wing expansion defects) and resembled those observed with knockdown of rk using RNAi. Flies that expressed CFP-tBur-&betal-α or rk RNAi ubiquitously generally reached pupal development however failed to eclose. Dead flies were fully developed but did not emerge from their pupal cases. Based on the parallel phenotypes with the RNAi flies, it was hypothesized that expression of CFP249 tBur-&betal-α resulted in a decrease of rk mediated signaling (Harwood, 2014).

Prior pharmacological studies have shown that sustained activation of a GPCR can trigger receptor desensitization, in turn leading to decreased receptor mediated signaling. Based on this precedent, it was postulated that CFP-tBur-&betal-α expression induces chronic stimulation of Rk in vivo resulting in receptor desensitization. The desensitized Rk receptor may not adequately respond to endogenous Bursicon at key developmental stages. To examine this possibility, in vitro studies were done comparing the effects of CFP-tBur-&betal-α and soluble Bursicon conditioned media on Rk activation/desensitization. Results show that long term stimulation of Rk (overnight) with either soluble or membrane tethered Bursicon renders the receptor unable to further respond to Bursicon. Notably, the desensitization of Rk is receptor specific. HEK 293 cells expressing recombinant Rk that has been desensitized to Bursicon can still signal in response to β2agonist treatment via endogenously expressed β2AR, which also signals through Gsα (Harwood, 2014).

Desensitization has previously been documented among other LGR receptors. For example, the human receptor LGR5 is known to be constitutively internalized and is one of the most evolutionarily related orthologs to rk (Snyder, 2013b). Notably, the C-terminal region of both Rk and LGR5 includes multiple serine residues, which, in the case of LGR5 has been shown to play an important role in desensitization (Snyder, 2013a). Taken together, it is proposed that tethered Bursicon mediated desensitization of Rk may underlie the loss of function phenotypes observed in vivo. Transgenic flies expressing tethered Bursicon under a UAS inducible promoter provide an important complementary genetic tool for studying the rickets Bursicon system (Harwood, 2014).

This study has shown that ubiquitous expression of CFP-tBur- β-α leads to developmental arrest at the pupal stage. Escapers that survive to adulthood show melanization and wing expansion phenotypes, features characteristic of rk knockdown. CFP-tBur- β-α induces phenotypes that are readily monitored, and provides a useful tool to further dissect the role of rk in the developing fly (complementing rk RNAi constructs) (Harwood, 2014).

During a previous screen performed in the Kopin lab, it was noted that RNAi mediated knockdown of the heterotrimeric G protein subunit Gαs, with muscle specific drivers resulted in developmental arrest (personal communication of Isabelle Draper to Harwood, 2014). It was hypothesized that Rk may be the receptor upstream of Gαs in muscle which led to this phenotype. To assess this possibility, CFP-tBur- &betal-α expression was restricted to muscle under the control of the pan mesodermal driver HOW-Gal4. CFP-tBur-&betal-α expression resulted in defects that phenocopied those seen with both ubiquitous expression of the tethered ligand and Gαs knockdown in muscle (i.e. lethality, wing phenotypes). Similar phenotypes were also observed when rk was downregulated using rk RNAi, confirming that the receptor was important in muscle. A series of Gal4 driver lines was used to target the tethered ligand to selected tissues and thus define the critical cell type(s) that require rk and underlie the lethality/wing expansion phenotypes. Although many studies have focused on the spatial and temporal release of Bursicon, the localization of the Bursicon receptors that are essential for survival, has remained elusive. This study strongly supports that a peripheral Rk, localized in muscle, plays an important role in Drosophila development (Harwood, 2014).

Expression of either CFP-tBur-&betal-α, or rk RNAi, in muscle leads to comparable lethality/wing phenotypes indicating that the receptor is present postsynaptically. In contrast, expression of rk RNAi in motor neurons (using the D42-Gal4 driver) has no effect, suggesting the absence of a presynaptic receptor. Expression of CFP-tBur- &betal-α using the same driver however leads to a wing defect phenotype. By design, CFP-tBur- &betal-α) anchors in the membrane and projects into the extracellular space (Harwood, 2013). Its function is defined primarily by three structural elements: transmembrane domain, linker, and peptide. It is postulated that tethered Bursicon anchored in the motor neuron may act on muscle Rk in trans leading to partial desensitization of the receptor thus inducing the wing expansion defect. In addition to defining a role for rk in muscle tissue, the role of rk during myogenesis was examined. It is well-established that during metamorphosis, most of the adult muscles are formed de novo from progenitor cells, i.e. adult muscle precursors (AMPs) located on the larval wing imaginal disc and leg imaginal disc. Undifferentiated AMPs express high levels of the helix-loop-helix transcription factor, Twist. As twist is downregulated, myoblasts commit to the muscle lineage and differentiate. This occurs in conjunction with an interplay of many transcription factors. A core regulatory network includes Tinman (cardiac muscle specification), Myocyte Enhancer Factor-2, Mef2 (which plays a key role in myoblast fusion/ formation of somatic muscles) and Apterous (which specifies selected subtypes of somatic muscles). Based on the above, selected drivers were used to target rickets at different stages of the myogenic process using either CFP-tBur- &betal-α or rk RNAi. Using the twist-gal4 driver this study has shown that downregulation of rk in AMPs had no effect on survival or wing expansion. This suggests that very early myogenesis is not altered by the absence of rk. In contrast it was shown that altering rk in differentiating myotubes (Mef2 positive cells) compromised survival and wing expansion (Harwood, 2014).

Bursicon and CCAP are co-packaged and released directly on to heart muscle. CCAP has an important role in modulating heart function. Given the coordinated synthesis and release of these two hormones, it was asked whether Rk mediated eclosion may also be linked to receptors expressed on Drosophila heart muscle. The analysis ruled out this possibility; decreased rk function in cardiac muscle did not compromise either eclosion or wing expansion (Harwood, 2014).

Whether expression of rk in selected subtypes of pharate adult muscles could account for the observed CFP-tBur-&betal-α/rk RNAi induced phenotypes was investigated. For these studies, the Act88F-Gal4 driver line was used that targets major muscles in the adult fly, including the indirect flight muscles. Expression of CFP-tBur-β-α or knockdown of rk in adult muscle results in developmental lethality prior to eclosion while escaper flies fail to expand their wings. Comparison of the tissues tissues that are targeted by the apterous Gal4 and Act88F Gal4 enabled exclusion of selected muscle subtypes potentially underlying the rk mediated phenotypes. Actin 88F is a muscle actin predominantly expressed in the thoracic indirect flight muscles (IFMs). Notably, apterous is absent in the IFMs, making this an unlikely target. Actin88F is also expressed in the mesothoracic leg (tibial depressor muscles), as well as in the abdominal muscles (both ventral and dorsal). These two muscle types thus emerge as potentially important for rk signaling-regulation of eclosion (Harwood, 2014).

It is well established that both leg and abdominal muscles are important during metamorphosis. At 12 hours after puparium formation, contraction of the abdominal muscles forces an air bubble forward which in turn, triggers head eversion. In addition, at the end of the pupal stage, the newly formed adult uses its legs to free itself from the case. Consistent with the hypothesis that rk is present in leg muscle, Bursicon immunoreactive neurons have been shown to directly innervate the leg of the cricket, Gryllus bimaculatus. The potential role of rk in leg muscle could also explain the kinked leg phenotype observed with rk classical mutants (Baker, 2005; Loveall, 2010; Harwood, 2014 and references therein).

In conclusion, a membrane tethered ligand (CFP-tBur-&betal-α) was developed that negatively regulates rk function when expressed in vivo. Using this novel tool in conjunction with existing RNAi fly lines adult muscle were identified as a tissue that requires rk expression for survival and wing expansion in Drosophila. In particular, based on this analysis it appears leg and abdominal muscles, or a subset of these, could be important. These studies set the stage for future investigations aimed at further understanding the role of muscle rk in fly development (Harwood, 2014).

Membrane tethered Bursicon constructs as heterodimeric modulators of the Drosophila G protein-coupled receptor rickets

The study of complex heterodimeric peptide ligands has been hampered by a paucity of pharmacological tools. To facilitate such investigations, this study explored the utility of membrane tethered ligands (MTLs). Feasibility of this recombinant approach was explored with a focus on Drosophila Bursicon, a heterodimeric cystine-knot protein that activates the G protein-coupled receptor Rickets (Rk). Rk/Bursicon signaling is an evolutionarily conserved pathway in insects required for wing expansion, cuticle hardening, and melanization during development. Two distinct MTL constructs were engineered, each composed of a type II transmembrane domain, a peptide linker, and a C terminal extracellular ligand that corresponded to either the α or β Bursicon subunit. Coexpression of the two complementary Bursicon MTLs triggered Rk-mediated signaling in vitro. Functionally active Bursicon MTLs were generated in which the two subunits were fused into a single heterodimeric peptide, oriented as either α-β or β-α. Carboxy-terminal deletion of 32 amino acids in the β-α MTL construct resulted in loss of agonist activity. Coexpression of this construct with rk inhibited receptor-mediated signaling by soluble Bursicon. This study has thus generated membrane-anchored Bursicon constructs that can activate or inhibit rk signaling. These probes can be used in future studies to explore the tissue and/or developmental stage-dependent effects of Bursicon in the genetically tractable Drosophila model organism. In addition, this success in generating functionally diverse Bursicon MTLs offers promise that such technology can be broadly applied to other complex ligands, including the family of mammalian cystine-knot proteins (Harwood, 2013).

Previously, only MTLs that included short peptide ligands (up to 39 amino acids) have been described. In contrast, the mature Bursicon subunits, α and β, are 141 and 121 amino acids, respectively. Furthermore, each of these subunits is a cystine-knot protein that includes a series of intramolecular disulfide bridges which confer tertiary structure. As an additional prerequisite of agonist activity, the α and β subunits must interact to form a structurally integrated heterodimer (Harwood, 2013).

Given the stringent requirements underlying the formation of active soluble Bursicon, including cellular coexpression, coprocessing, and cosecretion, the success in generating corresponding functional membrane tethered ligands could not have been anticipated. Initially, it was demonstrated that expression of both single tethered Bursicon subunits (α and β) in the same cell was sufficient to generate an active ligand. Follow-up studies revealed that coexpression of soluble and tethered complementary subunits also enabled the formation of active ligand. In contrast, when a single soluble subunit was added as conditioned media to cells expressing a tethered complementary subunit, no agonist activity was detectable. This finding suggests that intracellular assembly of the α-β heterodimer is a critical step in the formation of active hormone. These observations are consistent with reports on the heterodimerization requirements of soluble Bursicon and other cystine-knot proteins that are known to undergo intracellular assembly prior to secretion as an active ligand. Remarkably, both membrane tethered and soluble Bursicon subunits, despite the complexity of processing, appear to be fully compatible with each other in forming active heterodimers (Harwood, 2013).

In an attempt to further understand the structural requirements underlying tethered Bursicon function, constructs were generated in which both the α and β subunits were included in a single MTL. Since an active tethered ligand can be generated as either a β-α or α-β fusion construct, neither a free N nor a free C terminus is a requirement for agonist activity. It is noteworthy that conditioned medium containing a soluble form of the Bursicon fusion protein tested in the β-α arrangement also shows agonist activity. Whether tethered or soluble, the Bursicon fusions are active ligands. Observations with Bursicon reveal another parallel with mammalian heterodimeric cystine-knot proteins. Fusion of the α and β subunits of mammalian glycohormones including TSH, LH, and FSH as single soluble peptides also results in ligands that can activate their corresponding mammalian GPCR (Harwood, 2013).

The generation of tethered Bursicon fusion proteins provided a simplified model system to define domains of the dimer that are important for agonist activity. These experiments were guided by prior observations that the β subunit of mammalian glycohormones provides specificity and affinity for cognate receptors, whereas the α subunit is required for receptor activation. Furthermore, the literature suggests that the C terminal domain of the glycohormone α subunit is an important determinant for ligand activity. Based on this knowledge, a series of deletions were generated in the C terminus of Burs α in the context of the tBur-β-α heterodimer. These experiments demonstrated that the C terminal domain in tethered Bursicon was essential for Rk activation. One of the deletion mutants in which 32 C-terminal residues were truncated (designated as Δ32) not only led to loss of agonist activity, but also markedly inhibited the function of soluble Bursicon. This observation suggests that, once a domain essential for agonist activity is removed in the corresponding MTL, the remaining truncated peptide can inhibit soluble agonist–induced signaling. However, an MTL with a larger C terminal deletion (Δ35), although also lacking agonist activity, was much less effective (versus Δ32) in blocking soluble Bursicon–induced signaling. The difference between Δ32 and Δ35 is that three additional highly conserved residues including a critical cysteine are truncated in Δ35. The loss of these three residues may have compromised the tertiary structure of the tethered ligand, in turn explaining the functional difference in constructs. Soluble versions of the Δ32 and Δ35 constructs did not confer the same ability to block ligand-induced signaling. Thus, it is possible that membrane anchoring is required to generate a functional antagonist (Harwood, 2013).

It is of note that the GPCR targeted MTLs that had been reported prior to this study all shared a common orientation, in which the peptide ligand was expressed with a free extracellular N terminus. In contrast, the Bursicon MTLs were engineered with the opposite orientation (i.e., with a free extracellular carboxy terminus). This was achieved by incorporating a different transmembrane domain anchor (a type II TMD) into the construct. The ability to generate membrane-tethered ligands in either orientation markedly enhances the potential utility of MTL technology. For many peptides, orientation may be a critical factor in generating an active MTL. It is well established that, for peptide hormones recognizing class B GPCRs (e.g., secretin, parathyroid hormone, corticotropin releasing factor, glucagon-like peptide-1, gastric inhibitory polypeptide), the critical determinants of ligand efficacy reside in the N terminal domain of the hormone. Previous studies have shown that each of these peptides remains active when incorporated into an MTL that includes a type I TMD, i.e., the extracellular free end of the peptide is the N terminus. In contrast, peptide ligands recognizing class A GPCRs are more diverse. As examples, the amino termini of chemokines are generally considered critical for ligand activity, whereas for neuropeptides, functional determinants are often localized at the carboxyl terminus. In the latter case, it is anticipated that MTLs including a type II TMD will preserve biologic activity when corresponding peptides are anchored to the cell membrane (Harwood, 2013).

In summary, a strategy has been developed that can be widely applied to the study of peptide ligands. More specifically, Bursicon MTLs were identified that either activate or block Rk-mediated signaling. These findings set the stage for future in vivo studies. In the investigations to follow, tethered constructs will be selectively expressed in targeted tissues of Drosophila, thus exploring the utility of the approach for defining corresponding Rk-mediated pathways/physiologies. Precedent with these Bursicon MTLs will set the stage for parallel studies using other tethered cystine-knot proteins as tissue selective molecular probes. Candidate MTLs include mammalian glycohormones as well as non-GPCR regulators such as bone morphogenetic protein antagonists. The efficiency and flexibility of recombinant MTL technology will enable generation of a wide range of unique tools to complement the use of soluble ligands in understanding corresponding receptor-mediated physiologies (Harwood, 2013).

Bursicon functions within the Drosophila CNS to modulate wing expansion behavior, hormone secretion, and cell death

Hormones are often responsible for synchronizing somatic physiological changes with changes in behavior. Ecdysis (i.e., the shedding of the exoskeleton) in insects has served as a useful model for elucidating the molecular and cellular mechanisms of this synchronization, and has provided numerous insights into the hormonal coordination of body and behavior. An example in which the mechanisms have remained enigmatic is the neurohormone Bursicon, which, after the final molt, coordinates the plasticization and tanning of the initially folded wings with behaviors that drive wing expansion. The somatic effects of the hormone are governed by Bursicon that is released into the blood from neurons in the abdominal ganglion (the BAG), which die after wing expansion. How Bursicon induces the behavioral programs required for wing expansion, however, has remained unknown. This study shows by targeted suppression of excitability that a pair of Bursicon-immunoreactive neurons distinct from the BAG and located within the subesophageal ganglion in Drosophila (the BSEG) is involved in controlling wing expansion behaviors. Unlike the BAG, the BSEG arborize widely in the nervous system, including within the abdominal neuromeres, suggesting that, in addition to governing behavior, they also may modulate the BAG. Indeed, it was shown that animals lacking Bursicon receptor function have deficits both in the humoral release of Bursicon and in posteclosion apoptosis of the BAG. These results reveal novel neuromodulatory functions for Bursicon and support the hypothesis that the BSEG are essential for orchestrating both the behavioral and somatic processes underlying wing expansion (Peabody, 2008).

The study of insect ecdysis has provided a productive model for investigating the hormonal control of behavior. Bursicon acts directly after adult ecdysis in Drosophila to initiate the somatic processes and motor programs underlying wing expansion and to induce rapid cuticle tanning. In Drosophila,the architecture of Bursicon release differs from that of the other principal hormones that regulate ecdysis-related behaviors in that it is secreted not from a homogenous group of cells subject to common regulatory control, but instead from two distinct subsets of neurons. The work presented in this study demonstrates that this anatomical partition mirrors functional differences between the two subsets of Bursicon-expressing neurons. One group (the BAG) secretes Bursicon into the hemolymph, and the other (the BSEG) releases it widely in the nervous system to initiate the motor programs that underlie wing expansion. The observation that secretion of Bursicon from the BAG is impaired in rickets mutants is consistent with a model in which Bursicon secreted by the BSEG also modulates release from the BAG. These results thus provide a framework for understanding how the somatic actions of Bursicon are coordinated with its effects on behavior. In addition, the finding that apoptosis of the BAG is delayed in rickets mutants exposes a novel neuromodulatory function for Bursicon (Peabody, 2008).

That Bursicon is released into the blood from neurons in the abdominal ganglia has long been known from work in several insects and was recently confirmed in Drosophila. Segmentally represented pairs of cells homologous to the BAG also have been identified in abdominal neuromeres of the hawkmoth Manduca sexta and other insects, in which the anatomy of their projections closely resembles that described in this study. The large amount of Bursicon expressed in the axons of these neurons after they leave the CNS supports the conclusion that they are responsible for most, if not all, of the hormone released into the blood. Once in the blood, the hormone has been shown not only to activate tanning, in part by upregulating epidermal tyrosine hydroxylase, but also to alter the physiology of the wing. Early evidence that Bursicon plasticizes the cuticle of the wing before expansion in Manduca has been confirmed recently using recombinant hormone, and genetic evidence from Drosophila indicates that Bursicon mediates apoptosis of the wing epidermis after expansion as a prerequisite for the fusion of the two cuticular panels. Anatomical and functional evidence thus supports a humoral role for Bursicon released from the BAG, with these neurons mediating changes in the wing cuticle that support expansion. This conclusion is consistent with the projection pattern of these neurons described here, as well as with the observation that selective suppression of the BAG (i.e., in c929-Gal4>3× UAS-EKO animals) blocks wing expansion, even though this manipulation leaves expansional behaviors intact. Presumably the inhibition of Bursicon release into the hemolymph in these animals prevents the changes in cuticle plasticity required to render the wings pliable (Peabody, 2008).

The data presented in this study demonstrate that the Drosophila BSEG, and not the BAG, are required for wing expansion behaviors. The broad projection pattern of these neurons in the CNS is consistent with their targeting multiple motor systems to activate both air swallowing and abdominal contraction, although their precise targets remain to be determined. Work from Manduca suggests that the circuitry underlying wing expansion may be similar in this insect. Wing expansion in Manduca is known to require intact descending connections from the subesophageal ganglion, and hawkmoths have BSEG homologues, which localize to the labial neuromere of the subesophageal ganglion and send descending projections to all posterior ganglia. Further work will be required to determine the generality of this functional neuronal architecture in insects. Blowflies appear to represent an exception insofar as Bursicon has been reported absent in the subesophageal ganglion in these animals. Instead, Bursicon is synthesized by neurosecretory cells of the brain, which have been implicated in wing expansion behaviors. Reexamination of these conclusions, which predate the availability of antibodies to the hormone and were based on tanning bioactivity assays, should help clarify the extent to which Bursicon-expressing neurons in the subesophageal ganglion are likely to play a common role in insects (Peabody, 2008).

The widespread release of Bursicon in the nervous system, evidenced by the depletion of anti-Burs immunostaining from the BSEG fibers after eclosion, suggests that centrally secreted Bursicon has multiple functions. The discovery that rickets mutants, which lack a functional Bursicon receptor, exhibit diminished humoral release of Bursicon from the BAG points to a role beyond behavioral control. It remains to be shown that Bursicon acts as a centrally derived paracrine factor in potentiating its release from the BAG, but there is reason to believe that the BAG may be direct targets of Bursicon. Bursicon signaling is mediated by the cAMP pathway, and it has been shown that suppression of protein kinase A (PKA), one of the principal effectors of this pathway, decreases the release of Bursicon (Luan, 2006). Although this reduction was not sufficient to disrupt wing expansion and tanning, more recent experiments demonstrate that greater suppression of PKA reduces Bursicon release sufficiently to cause highly penetrant wing expansion deficits. This is consistent with the observations of Zhao (2008), who similarly reported that overexpression of a cAMP phosphodiesterase (UAS-dunce) in the BAG using c929-Gal4 results in wing expansion deficits in many flies when two copies of UAS-dunce are expressed (Peabody, 2008).

The conclusion that centrally secreted Bursicon participates in regulating its release as a hormone from the BAG is interesting in light of the long-standing observation that Bursicon release into the hemolymph requires descending signals from the head. Decapitation or neck ligation of both blowflies and Drosophila soon after eclosion prevents tanning and wing expansion. Incisions that sever the ventral nerves in the neck of blowflies have also been reported to prevent tanning, but not air swallowing, suggesting that air swallowing, like tanning, is initiated by a signal that originates in the head. Although it is unclear that the circuitry governing Bursicon release is conserved in both types of fly, this observation is consistent with a mechanism in which Bursicon secreted by the BSEG acts within the subesophageal ganglion or brain to initiate air ingestion and as a descending signal to promote Bursicon secretion from the BAG. Since disruption of the Bursicon signaling pathway only partially attenuates Bursicon release from the BAG, as reported previously Baker (2002), other regulatory signals must also exist (Peabody, 2008).

The discovery that the apoptosis of BAG neurons is delayed in rickets mutants indicates that Bursicon promotes cell death in the CNS, as it does in the wing. Apoptosis of the BAG and other CCAP-expressing neurons in the ventral ganglia is regulated by declining titers of 20-hydroxyecdysone (20E), which induces expression of the cell death genes reaper and hid. Bursicon presumably facilitates one or more steps in this process. Promotion of apoptosis may, in fact, be a general role of Bursicon in the CNS, given that many neurons in the thoracic and abdominal ganglia have been shown to die after eclosion in response to a head-derived signal, the release of which correlates closely with wing expansion. Further work will be required to test this hypothesis, but since the circuitry and musculature required for molting is substantially eliminated after eclosion, it would be parsimonious if Bursicon, which mediates the final physiological events of the terminal molt, were also to facilitate the demise of this machinery (Peabody, 2008).

In summary, these results provide key insights into the cellular mechanisms underlying Bursicon's regulation of wing expansion in Drosophila. Because wing expansion follows adult ecdysis, Bursicon's release is almost certainly modulated by the hormones that govern the events underlying that process. A remaining challenge is thus to understand the integrative mechanisms that coordinate the release of Bursicon with that of ecdysis-related hormones, such as ecdysis-triggering hormone and eclosion hormone. The functional architecture of the Bursicon system described here should inform these investigations and help elucidate the cellular circuitry that more generally mediates somatic and behavioral coordination during the ecdysis and postecdysis phases. These results have demonstrated that Bursicon contributes more globally to postecdysis than originally imagined (Peabody, 2008).

Mutations in the Drosophila glycoprotein hormone receptor, rickets, eliminate neuropeptide-induced tanning and selectively block a stereotyped behavioral program

Adult insects achieve their final form shortly after adult eclosion by the combined effects of specialized behaviors that generate increased blood pressure, which causes cuticular expansion, and hormones, which plasticize and then tan the cuticle. This study examined the molecular mechanisms contributing to these processes in Drosophila by analyzing mutants for the rickets gene. These flies fail to initiate the behavioral and tanning processes that normally follow ecdysis. Sequencing of rickets mutants and STS mapping of deficiencies confirmed that rickets encodes the glycoprotein hormone receptor DLGR2. Although rickets mutants produce and release the insect-tanning hormone Bursicon, they do not melanize when injected with extracts containing Bursicon. In contrast, mutants do melanize in response to injection of an analog of cyclic AMP, the second messenger for Bursicon. Hence, rickets appears to encode a component of the Bursicon response pathway, probably the Bursicon receptor itself. Mutants also have a behavioral deficit in that they fail to initiate the behavioral program for wing expansion. A set of decapitation experiments utilizing rickets mutants and flies that lack cells containing the neuropeptide eclosion hormone, reveals a multicomponent control to the activation of this behavioral program (Baker, 2002).

In mammals, mutations in glycoprotein hormone receptors cause developmental defects due to endocrine misregulation. Drosophila has three identified members of this family of G-protein-coupled receptors. The gene of one of these, DLGR2, has been ascribed to the rickets mutation. The data support the conclusion that rk mutations arise from changes in the DLGR2 gene. STS analysis showed that deletions that had been genetically defined as breaking within the rk gene result in a partial removal of the DLGR2 transcription unit. Also, sequencing of two strong mutant alleles of rk showed that these strains had mutations that resulted in premature stop codons in the critical transmembrane domain of the receptor. Hence, this study agrees with the suggestion that rk mutations arise from disruption of the DLGR2 gene (Baker, 2002).

A point of controversy, however, comes from an analysis of the phenotype that arises from the removal of the rk product. Eriksen (2000) described the sequence and expression profile of DLGR2 and showed that flies carrying a p-element inserted into the 5′-untranslated region of DLGR2 had an embryonic lethal phenotype. Their paper did not, however, address the synonymy of DLGR2 and rk or the fact that out of 22 reported rk alleles, rkw11p is the only one that is lethal. The data presented in the current study indicate that complete removal of the rk function is not lethal. Flies that carry deficiencies that truncate DLGR2 from telomeric and centromeric directions are unlikely to make functional protein, but are viable and fertile with only a failure of tanning and post-ecdysial expansion. Two strong alleles, rk1 and rk4, are shown to contain nonsense mutations in the transmembrane containing exon and these also show the classic rk phenotype. Moreover, the phenotype is the same when these mutations are over deficiencies for the region. The embryonic lethality observed in the rkw11p stock seems more likely to be due to the presence of an unidentified lethal mutation elsewhere on the second chromosome, or to an effect of misexpressing DLGR2 in embryonic tissues in a way that is lethal. The observation that the lethality and the p-element insert are separable by recombination supports the second-site lethal interpretation for the embryonic lethality. Also, crosses of rkw11p with either Df(2L)b-L or Df(2L)A376 give a rk phenotype but no embryonic lethality. From the loss of function phenotypes, it is concluded that normal DLGR2 function is associated only with signaling processes that occur around the time of ecdysis (Baker, 2002).

The rk mutations specifically interfere with the last phase (the expansional phase) of the ecdysis sequence. These flies fail to expand their wings and thorax and they delay the onset of tanning and melanization. This phenotype is also observed in flies that have had their eclosion hormone (EH) cells genetically killed, implicating EH in the regulation of post-ecdysial expansional behavior as well as of ecdysis itself. This analysis of the EH system in rk flies, however, shows that they respond normally by initiating ecdysis when challenged with MasETH, a response that requires the activity of the EH neurons, and that they show EH depletion from the nervous system following eclosion. These results indicate that rk is probably downstream of EH release and action (Baker, 2002).

In blowflies and Drosophila decapitation immediately after ecdysis results in a delay in tanning that phenocopies rk mutations. As with the rk flies, such decapitated flies gradually darken over a period of several hours after decapitation. The lack of rapid pigmentation has been ascribed to the lack of the tanning hormone Bursicon. The assay of blood from rk flies shortly after emergence shows that these flies contain Bursicon activity and that they release it on schedule. The quantitative difference in Bursicon activity between rk and wild type is probably due to differences in the timing; no behavioral assay for the time of hormone release is available in the mutants and, therefore, it was necessary to rely on time after eclosion. However, rk flies do not respond to injection of Bursicon-containing material; neither the blood from normal Drosophila that are expanding their wings nor CNS extracts from the fly Sarcophaga provoked a response. Although these flies do not melanize in response to Bursicon extracts, they show prominent melanization in response to injection of cAMP, the suggested second messenger mediating Bursicon action. This result shows that the cuticle of rk flies has all of the machinery needed for melanization, but that it simply cannot activate this machinery in response to Bursicon. It also argues that the lesion caused by rk probably occurs in the Bursicon signaling pathway between the reception of Bursicon and the production of cAMP. From the nature of the DLGR2 product, it is thought that the most likely possibility is that it is the receptor for Bursicon. The large extracellular domain of this family of receptors is used for binding large, glycoprotein hormones. Intriguingly, Bursicon is one of the largest of the insect hormones, with an estimated molecular mass of 33 kDa. Because of its size, however, this hormone has yet to be isolated and sequenced. Therefore, it is not yet possible to test directly whether DLGR2 can bind Bursicon (Baker, 2002).

Hormonal and behavioral control over wing expansion in Drosophila is time dependant and appears to involve both EH and rk (and, by inference, Bursicon). In wild-type individuals, decapitation within 10 min of ecdysis prevents both the release of Bursicon (shown by their lack of rapid tanning) and wing expansion. During a transition period starting at around 10 min post-ecdysis, decapitated flies would occasionally tan without wing expansion, but not vice versa. Shortly thereafter, both tanning and wing expansion consistently followed decapitation. This timing suggests that Bursicon release and wing expansion are activated at about 10 min post-ecdysis in a stereotypical sequence, with Bursicon release being followed by activation of the wing expansion program. This pattern is consistent with a model in which Bursicon release triggers the wing expansion motor programs. This study found that in rk mutants, the release of Bursicon occurs on schedule but the expansional behaviors fail to occur. If rk does indeed encode the Bursicon receptor, then this is the first direct evidence that Bursicon activates the wing expansion program (Baker, 2002).

In Drosophila, the effects of post-ecdysial ligatures mirror those seen in the blowflies Sarcophaga and Calliphora. Surprisingly, this study has identified a pre-ecdysial period during which ligation activates the post-ecdysial behavior and melanization rather than inhibiting it. There is an early response window that begins when the animals reach the 'grainy stage' approximately 3 h before ecdysis. Pharate adults never eclose if decapitated prior to this time but routinely eclose if decapitated after they start the grainy stage. The latencies from decapitation to ecdysis ranged from 20 min to 5 h, with some flies emerging earlier than expected, based on their developmental stage, and other flies emerging later. None of the late emerging flies were seen to then tan or spread their wings, but, by contrast, most of the prematurely ecdysing individuals showed normal tanning and wing expansion despite lacking their head. Interestingly, these flies began wing expansion during or immediately after ecdysis rather than waiting the 10-15 min that is typical for intact animals. The significance of this difference in timing will be discussed below. During this early window, between 3 h and 50 min before ecdysis (the start of the 'extended ptilinum stage'), the fraction of flies showing precocious ecdysis and wing expansion gradually increased with time. A different set of responses were observed starting at about 50 min before ecdysis. During this second window, the latency period before ecdysis, after decapitation, was abruptly reduced down to about a minute or less, but tanning and wing expansion never occurred. Therefore, in terms of wing expansion, flies decapitated during this second window behaved in the same way as flies decapitated immediately after eclosion (Baker, 2002).

Two pieces of evidence link these response windows to EH. First, the transitions in the response of developing flies to decapitation correlate with immunocytochemical changes in the EH-expressing cells. At 4-7 h before ecdysis there is a shift in EH immunostaining that represents either an initial release of EH or a redistribution of EH to the axon terminals. At around 50 min before ecdysis the major depletion of the EH neurons occurs. Second, flies that lack EH neurons do not show premature ecdysis in response to decapitation, and they do not subsequently tan or expand their wings. In contrast, experiments with rk flies suggest that Bursicon is not involved in the early window. When decapitated during the initial window, early ecdysing mutant flies also expand their wings, although the wings subsequently collapse because tanning does not occur. Hence, the elements driving the wing expansion program are present in the mutant but cannot be activated in response to Bursicon after eclosion (Baker, 2002).

These data, coupled with findings that flies decapitated prior to adult ecdysis often spread their wings, suggest that an inhibitory component descending from the brain/SEG suppresses these ecdysial and post-ecdysial motor programs. At about 3 h before ecdysis developmental changes make the ecdysis and post-ecdysis motor programs competent to be expressed. The data from the EH cell knockout flies suggest that the EH neurons are involved with this competence, and it may be a low-level release of EH that initially activates the motor programs. Although expansion occurs along with a response to Bursicon in this case, it does not depend on it as shown by the ability of rk flies to expand their wings if decapitated at this early time. The major release of EH at 50 min before ecdysis causes a strong activation of the ecdysis program but suppresses both Bursicon secretion and the wing expansion program. Decapitation after this EH release is rapidly followed by ecdysis but wing expansion is never displayed. Subsequent descending commands from the head that occur after ecdysis bring about Bursicon release and this, in turn, is required to activate the expansional program (Baker, 2002).

This seemingly complex control may have arisen from elaboration of a simpler control system evident in most other insects. In many insects ecdysis and tanning are closely linked and expansion begins as the insect is escaping from the old cuticle. By contrast, insects that pupate underground or in confined sites delay the expansion of delicate wings until the insect has dug its way to freedom. In these insects, the fixed relationship between the ecdysial and expansional phases has been replaced by a period of behavioral flexibility that allows escape from a buried pupation chamber before the initiation of cuticular expansion and hardening. This separation is found in the Cycloraphous Diptera and also in some months such as the tobacco hornworm, Manduca sexta (Baker, 2002).

Intriguingly, during larval ecdysis of Manduca and Drosophila, expansion is already underway at the start of ecdysis. Hence, in these early stages both programs may be under the direct activation of EH. This link between ecdysis and expansion must be reconfigured during adult development to give the delay between ecdysis and expansion that is characteristic of adult behavior. It appears that in Drosophila, the larval relationships may remain intact but are masked by a strong inhibition that is imposed at the time of the major release of EH, about 50 min before ecdysis. Decapitation prior to this time may reveal the persisting larval circuit that links the ecdysial and post-ecdysial phases under common control of EH (Baker, 2002).

In most insects, the activation of ecdysis and tanning are linked to limit the risk of desiccation and predation to the soft, flexible post-ecdysis cuticle. In Drosophila and Manduca, evolution has replaced this program with a period of behavioral flexibility that permits the adult insect to take advantage of sensory information to escape its pupal confinement and to control the time and place in which the expansional behaviors are initiated (Baker, 2002).


REFERENCES

Search PubMed for articles about Drosophila Rickets

Baker, J. D. and Truman, J. W. (2002). Mutations in the Drosophila glycoprotein hormone receptor, rickets, eliminate neuropeptide-induced tanning and selectively block a stereotyped behavioral program. J Exp Biol 205: 2555-2565. PubMed ID: 12151362

Baker, P. W., Tanaka, K. K., Klitgord, N. and Cripps, R. M. (2005). Adult myogenesis in Drosophila melanogaster can proceed independently of myocyte enhancer factor-2. Genetics 170: 1747-1759. PubMed ID: 15956678

Diao, F. and White, B. H. (2012). A novel approach for directing transgene expression in Drosophila: T2A-Gal4 in-frame fusion. Genetics 190: 1139-1144. PubMed ID: 22209908

Eriksen, K. K., Hauser, F., Schiott, M., Pedersen, K. M., Sondergaard, L. and Grimmelikhuijzen, C. J. (2000). Molecular cloning, genomic organization, developmental regulation, and a knock-out mutant of a novel leu-rich repeats-containing G protein-coupled receptor (DLGR-2) from Drosophila melanogaster. Genome Res 10: 924-938. PubMed ID: 10899142

Harwood, B. N., Fortin, J. P., Gao, K., Chen, C., Beinborn, M. and Kopin, A. S. (2013). Membrane tethered Bursicon constructs as heterodimeric modulators of the Drosophila G protein-coupled receptor rickets. Mol Pharmacol 83: 814-821. PubMed ID: 23340494

Harwood, B. N., Draper, I. and Kopin, A. S. (2014). Targeted inactivation of the rickets receptor in muscle compromises Drosophila viability. J Exp Biol 217(Pt 22):4091-8. PubMed ID: 25278473

Honegger, H. W., Dewey, E. M. and Ewer, J. (2008). Bursicon, the tanning hormone of insects: recent advances following the discovery of its molecular identity. J Comp Physiol A Neuroethol Sens Neural Behav Physiol 194: 989-1005. PubMed ID: 19005656

Lahr, E. C., Dean, D. and Ewer, J. (2012). Genetic analysis of ecdysis behavior in Drosophila reveals partially overlapping functions of two unrelated neuropeptides. J Neurosci 32: 6819-6829. PubMed ID: 22593051

Loveall, B. J. and Deitcher, D. L. (2010). The essential role of Bursicon during Drosophila development. BMC Dev Biol 10: 92. PubMed ID: 20807433

Peabody, N. C., Diao, F., Luan, H., Wang, H., Dewey, E. M., Honegger, H. W. and White, B. H. (2008). Bursicon functions within the Drosophila CNS to modulate wing expansion behavior, hormone secretion, and cell death. J Neurosci 28: 14379-14391. PubMed ID: 19118171

Peabody, N. C., Pohl, J. B., Diao, F., Vreede, A. P., Sandstrom, D. J., Wang, H., Zelensky, P. K. and White, B. H. (2009). Characterization of the decision network for wing expansion in Drosophila using targeted expression of the TRPM8 channel. J Neurosci 29: 3343-3353. PubMed ID: 19295141

Snyder, J. C., Rochelle, L. K., Lyerly, H. K., Caron, M. G. and Barak, L. S. (2013a). Constitutive internalization of the leucine-rich G protein-coupled receptor-5 (LGR5) to the trans-Golgi network. J Biol Chem 288: 10286-10297. PubMed ID: 23439653

Snyder, J. C., Rochelle, L. K., Barak, L. S. and Caron, M. G. (2013b). The stem cell-expressed receptor Lgr5 possesses canonical and functionally active molecular determinants critical to beta-arrestin-2 recruitment. PLoS One 8: e84476. PubMed ID: 24386388

Zhao, T., Gu, T., Rice, H. C., McAdams, K. L., Roark, K. M., Lawson, K., Gauthier, S. A., Reagan, K. L. and Hewes, R. S. (2008). A Drosophila gain-of-function screen for candidate genes involved in steroid-dependent neuroendocrine cell remodeling. Genetics 178: 883-901. PubMed ID: 18245346


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date revised: 15 November 2014

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