Silencing D. melanogaster lgr1 impairs transition from larval to pupal stage
Introduction
Insects, which constitute over 80% of all animal species, have a huge impact on our environment and agriculture. A number of insects are infamous pest species or disease vectors, while others are very beneficial, for example as pollinators of flowering plants. Even though at first glance mammals and insects are very little alike, molecular genetic and physiological studies over the past years showed that fundamental aspects of many biological processes are well conserved throughout evolution.
In vertebrate physiology, the glycoprotein hormones are a group of key regulatory compounds. These heterodimeric proteins consist of an identical α-subunit in combination with a hormone-specific β-subunit. Follicle stimulating hormone (FSH) is required in the maturation and maintenance of germ cells in both males and females, but it is also a determining factor for bone mass and for the growth of tumour blood vessels (Allan et al., 2010; Laan et al., 2012, Radu et al., 2010, Sun et al., 2006). Thyroid stimulating hormone (TSH) is produced in the pituitary gland and induces the growth of thyroid follicular cells and the release of thyroid hormones which are important regulators of metabolism (Gogakos et al., 2010) and play a crucial role in the regulation of metamorphosis (Laudet, 2011). Luteinising hormone (LH) is responsible for the regulation of testosterone production in the male’s Leydig cells (Chen et al., 2009). In females, it acts as the trigger for ovulation and is responsible for the formation of the corpus luteum which secretes progesterone. During pregnancy, the maintenance of the corpus luteum is taken over by human chorionic gonadotropin (hCG) which binds to the same receptor as LH and thus ensures the continued production of progesterone (Cole, 2010).
The receptors for the glycoprotein hormones are members of the leucine-rich repeats containing G protein-coupled receptors (LGRs). Like all rhodopsin-type GPCRs, these receptors have a membrane-spanning domain consisting of seven α-helices but their characteristically large ectodomain is their hallmark feature. This ectodomain holds the leucine-rich repeats that assume a horseshoe-shaped conformation and contain residues that are essential for the receptor’s specificity (Kajava et al., 1995, Smits et al., 2003). Furthermore, these receptors feature a hinge region linking the ectodomain to the transmembrane domain. The hinge region has long been largely uncharacterised but recently, its structure was determined for the FSH receptor and it was predicted to play a key role in the activation of this receptor (Jiang et al., 2012).
The LGRs appear to be remarkably well conserved, with LGRs having been identified in Placozoa, Cnidaria, Nematoda, Arthropoda, Mollusca, Hemichordata, Echinodermata and Chordata. The family of LGRs can be divided into three major types based on the number of leucine-rich repeats (LRRs) in the receptor’s ectodomain, their hinge region and the presence or absence of an LDLa motif. Type A LGRs display 7–9 LRRs and are characterised by a long hinge region whereas LGRs of the type B feature twice as many LRRs (16–18) and have a shorter hinge region (but still larger than in type C LGRs). The hallmark of the type C LGRs is the presence of one or more LDLa motifs. The number of LDLa motifs allows for a further division of type C LGRs into two subtypes: a C1 type containing only one LDLa, which is well-conserved throughout many phyla, and a C2 type containing multiple LDLa motifs and having a distinct hinge region. This latter type of LGRs is rarer but has been identified in echinoderms and mollusks, as well as in Pediculus humanis corporis. Interesting with regards to the origin of LGRs are eight putative LGRs that were found in Trichoplax and which resemble type-C LGRs, but lack the LDLa motif. It has been proposed that these receptors might represent an ancient form of the type-C LGRs, prior to the incorporation of the LDLa motif, but it cannot be ruled out that Trichoplax LGR has lost its LDLa motif during the course of evolution (Van Hiel et al., 2012).
Although for invertebrate LGRs little evidence has currently been gathered on their biological functions, the important roles of their mammalian homologues and their evolutionary conservation suggest that they may be essential in these animals as well. So far, the only invertebrate LGR that was characterised extensively is LGR2 from D. melanogaster. This is the receptor for the heterodimeric neurohormone bursicon, which induces hardening and tanning of the cuticle and pupal case (Loveall and Deitcher, 2010, Luo et al., 2005, Mendive et al., 2005). Additionally, the hormone also co-ordinates the maturation of the wings in newly eclosed adult flies (Kimura et al., 2004, Natzle et al., 2008, Peabody et al., 2008).
In this paper, we have studied the closest fruit fly homologue of the vertebrate glycoprotein hormone receptors, D. melanogaster LGR1 (CG7665). This receptor, which was cloned in 1997 (Hauser et al., 1997), can be activated by the heterodimer GPA2/GPB5 (Sudo et al., 2005). Both subunits are cystine knot proteins that show similarities with the glycoprotein hormone subunits α and β, respectively. GPA2 and GPB5 were identified in the fruit fly based on their homology with the subunits of thyrostimulin, a hormone that was discovered to activate the vertebrate TSH receptor (Nakabayashi et al., 2002). Recent in situ hybridisation data showed expression of GPA2 and GPB5 in the same cells of the third instar CNS and using a GPB5-Gal4 line, the β-subunit producing cells were localised in larval and adult fruit flies (Sellami et al., 2011). Moreover, this study also pointed at the possible role of GPA2/GPB5 as an antidiuretic factor, while ablation of neurons producing this LGR1-ligand resulted in a developmental defect that led to reduced numbers of adult flies. In line with this hypothesis, GPA2/GPB5 was recently shown to affect the transport of Na+ and K+ across the hindgut of the mosquito, Aedes aegypti (Paluzzi et al., 2014).
In the present paper, we report on transcript profiling studies that were performed for D. melanogaster lgr1 and for the genes encoding the hormone subunits, gpa2 (CG17878) and gpb5 (CG40041). Moreover, by means of an RNA interference (RNAi) construct targeting lgr1, we identified a remarkable developmental phenotype in D. melanogaster.
Section snippets
Phylogenetic analysis
Multiple sequence alignment was performed in MAFFT (L-INS-i method) (Katoh et al., 2005, Katoh et al., 2002) and converted to MEGA 5.10 (Tamura et al., 2011) wherein genetic distances between sequences were calculated and both a Maximum-Likelihood and a Neighbour-Joining tree were constructed. The Maximum-Likelihood tree was created using the Jones-Taylor-Thornton amino acid substitution model. The reliability of the tree was estimated by 2000-fold bootstrap re-sampling. The Neighbour-Joining
Phylogenetic analysis of well-described LGR sequences
A phylogenetic analysis was performed with a limited set of well-annotated LGRs from different animal phyla using Maximum-Likelihood (Fig. 1) and Neighbour Joining (Fig. 2) analysis. In both trees, the three main types of LGRs (A-, B-, C-type) are indeed situated in distinct clusters. A schematic representation of the overall structural organization of the LGR-types can be found in Supplementary Fig. 1A, along with the multiple sequence alignment used for the creation of the phylogenetic trees.
Phylogenetic analysis of well-described LGR sequences
The occurrence of LGRs from Cnidaria and Nematoda within the A-type cluster, to which D. melanogaster LGR1 belongs as well, may suggest that this LGR-type is the more ancient one (Fig. 1 and Fig. 2). However, relatively frequent gene losses or gene multiplications can be observed in different metazoan evolutionary lineages. An interesting example of LGR gene duplication in insects are the two type A LGRs that are encoded in Tribolium castaneum, making this beetle the only invertebrate so far to
Conclusion
In this paper, we have elucidated the developmental and tissue distribution of the transcripts for lgr1 and for the genes encoding the glycoprotein hormone related subunits, gpa2 and gpb5. Furthermore, we have described the phenotype associated with lgr1 transcript silencing. This included a prolonged third instar stage and a developmental arrest as third instar or early pupa. The reduction of the transcript levels of the Halloween genes sad and spok as well as of the genes sgs4 and ilp6
Acknowledgments
The authors gratefully acknowledge M.B. O’Connor and L. Riddiford for the kindly donated ring gland-specific Gal4-driver lines and J.-P. Delbecque for the kindly provided antibody and tracer solutions for ecdysteroid quantification. They also thank J. Van Duppen for excellent technical assistance. Funding for this study was provided by the Interuniversity Attraction Poles program [Belgian Science Policy Grant (P7/40)], the Flemish (FWO) and the KU Leuven (GOA/11/02) Research Foundation. HPV was
References (70)
- et al.
Glue secretion in the Drosophila salivary gland: a model for steroid-regulated exocytosis
Dev. Biol.
(2001) - et al.
Ras activity in the Drosophila prothoracic gland regulates body size and developmental rate via ecdysone release
Curr. Biol.
(2005) - et al.
Leydig cells: from stem cells to aging
Mol. Cell. Endocrinol.
(2009) - et al.
Enteric neurons and systemic signals couple nutritional and reproductive status with intestinal homeostasis
Cell Metab.
(2011) - et al.
Thyroid and bone
Arch. Biochem. Biophys.
(2010) - et al.
Molecular cloning, genomic organization, and developmental regulation of a novel receptor from Drosophila melanogaster structurally related to members of the thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone/choriogonadotropin receptor family from mammals
J. Biol. Chem.
(1997) - et al.
Modeling of the three-dimensional structure of proteins with the typical leucine-rich repeats
Structure
(1995) The variation of blood volume with age in the desert locust (Schistocerca gregaria Forsk.)
J. Insect Physiol.
(1961)- et al.
Control of ecdysteroidogenesis in prothoracic glands of insects: a review
Peptides
(2010) - et al.
Role of the halloween genes, spook and phantom in ecdysteroidogenesis in the desert locust, Schistocerca gregaria
J. Insect Physiol.
(2011)
Prothoracicotropic hormone regulates developmental timing and body size in Drosophila
Dev. Cell
Drosophila molting neurohormone bursicon is a heterodimer and the natural agonist of the orphan receptor DLGR2
FEBS Lett.
Hormonal control of tanning in the American cockroach: changes in blood cell permeability during ecdysis
J. Insect Physiol.
The role of the prothoracic gland in determining critical weight for metamorphosis in Drosophila melanogaster
Curr. Biol.
A fat body-derived IGF-like peptide regulates postfeeding growth in Drosophila
Dev. Cell
Spook and spookier code for stage-specific components of the ecdysone biosynthetic pathway in Diptera
Dev. Biol.
Conversion of 3-oxo steroids into ecdysteroids triggers molting and expression of 20E-inducible genes in Drosophila melanogaster
Biochem. Biophys. Res. Commun.
Neuroendocrine cells in Drosophila melanogaster producing GPA2/GPB5, a hormone with homology to LH, FSH and TSH
Gen. Comp. Endocrinol.
A Drosophila insulin-like peptide promotes growth during nonfeeding states
Dev. Cell
FSH directly regulates bone mass
Cell
Thyrostimulin, but not thyroid-stimulating hormone (TSH), acts as a paracrine regulator to activate the TSH receptor in mammalian ovary
J. Biol. Chem.
An evolutionary comparison of leucine-rich repeat containing G protein-coupled receptors reveals a novel LGR subtype
Peptides
Studies on the Black Box: incorporation of 3-oxo-7-dehydrocholesterol into ecdysteroids by Drosophila melanogaster and Manduca sexta
Insect Biochem. Mol. Biol.
Phantom encodes the 25-hydroxylase of Drosophila melanogaster and Bombyx mori: a P450 enzyme critical in ecdysone biosynthesis
Insect Biochem. Mol. Biol.
Neuroendocrine regulation of ecdysis
Follicle-stimulating hormone increases bone mass in female mice
Proc. Natl. Acad. Sci. USA
Insect neuropeptide bursicon homodimers induce innate immune and stress genes during molting by activating the NF-kappaB transcription factor Relish
PLoS One
Large-scale RNAi screen of G protein-coupled receptors involved in larval growth, molting and metamorphosis in the red flour beetle
BMC Genomics
Using FlyAtlas to identify better Drosophila melanogaster models of human disease
Nat. Genet.
A remarkable, precisely timed release of hyperglycemic hormone from endocrine cells in the gut is associated with ecdysis in the crab Carcinus maenas
Proc. Natl. Acad. Sci. USA
Biological functions of hCG and hCG-related molecules
Reprod. Biol. Endocrinol.
New discoveries on the biology and detection of human chorionic gonadotropin
Reprod. Biol. Endocrinol.
Antagonistic actions of ecdysone and insulins determine final size in Drosophila
Science
Secreted peptide Dilp8 coordinates Drosophila tissue growth with developmental timing
Science
Distinct expression patterns of glycoprotein hormone-alpha2 and -beta5 in a basal chordate suggest independent developmental functions
Endocrinology
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2024, General and Comparative EndocrinologyAncestral glycoprotein hormone and its cognate receptor present in primitive chordate ascidian: Molecular identification and functional characterization
2023, International Journal of Biological MacromoleculesCitation Excerpt :Unlike other GPHs which are restricted to vertebrates, GPH (GPA2/GPB5) has also been identified and characterized in invertebrate species, such as cephalochordates [13,14], insects [15,16], and nematodes [17]. An increasing number of studies have confirmed that the function of GPH (GPA2/GPB5), beyond the effect of thyroxine production, is involved in regulating reproduction [18], bone formation [19], immune response [20], and development [21,22] in many vertebrates and invertebrates. The comparative genomic analyses indicated that GPA2/GPB5 heterodimer might represent the ancestral version of vertebrate GPHs [23,24].
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2022, Comparative Biochemistry and Physiology - Part D: Genomics and ProteomicsCitation Excerpt :Considering that molting and energy metabolism are controlled by the ecdysteroid hormone in most arthropods (Shyamal et al., 2018), LGR1 is likely involved in these signaling systems. For instance, RNAi-mediated knockdown of LGR1 led to severe inhibition of white pupa formation in D. melanogaster and inhibition of expression of genes involved in ecdysteroid signaling (Vandersmissen et al., 2014). However, a detailed functional study in D. magna is required for clarification.
Exploring the role of glycoprotein hormone GPA2/GPB5 in the medically important insect, Rhodnius prolixus
2022, PeptidesCitation Excerpt :In the fruit fly Drosophila melanogaster, GPA2/GPB5 receptor (DLGR1) transcripts are highly expressed in the hindgut (HG), salivary glands (SGs), Malpighian tubules (MTs), and tissues with water transporting epithelia [19]. Thus, DLGR1 knockdown flies were shown to be less tolerant to dehydration stress, resulting in a significant reduction of water content in flies tested under desiccating conditions [20]. In addition, downregulation of DLGR1 resulted in developmental defects including suppression of white pupa formation in D. melanogaster larvae.
Functional role of the heterodimeric glycoprotein hormone, GPA2/GPB5, and its receptor, LGR1: An invertebrate perspective
2016, General and Comparative EndocrinologyCitation Excerpt :Though not as intensely stained, expression of both subunits was also observed in cells localized within other regions of the CNS including the pars intercerebralis, suboesophageal ganglion and thoracic ganglion (Sellami et al., 2011). GPA2 transcripts are also greater in the developing and mature testes of male flies compared to female gonads, whereas GPB5 transcripts were more prominent in female reproductive tissues (Vandersmissen et al., 2014). Based on the transcript expression profiles of the glycoprotein hormone subunits and LGR1, researchers have sought to identify potential developmental effects using RNA interference techniques (Vandersmissen et al., 2014).