Sesquiterpene action, and morphogenetic signaling through the ortholog of retinoid X receptor, in higher Diptera

General and Comparative Endocrinology 194 (2013) 326–335

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Review

Sesquiterpene action, and morphogenetic signaling through the ortholog of retinoid X receptor, in higher Diptera Davy Jones a,⇑, Grace Jones b,⇑, Peter E.A. Teal c a

Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40504, USA Department of Biology, University of Kentucky, Lexington, KY 40504, USA c USDA/ARS, Chemistry Research Unit, Gainesville, FL 32608, USA b

a r t i c l e

i n f o

Article history: Received 11 June 2013 Revised 11 September 2013 Accepted 29 September 2013 Available online 8 October 2013 Keywords: Ultraspiracle Methyl farnesoate RXR Retinoic acid Juvenile hormone Metamorphosis

a b s t r a c t Morphogenetic signaling by small terpenoid hormones is a common feature of both vertebrate and invertebrate development. Most attention on insect developmental signaling by small terpenoids has focused on signaling by juvenile hormone through bHLH-PAS proteins (e.g., the MET protein), especially as that signaling axis intersects with ecdysteroid action through the receptor EcR. However, a series of endocrine and pharmacological studies on pupariation in cyclorrhaphous Diptera have remained persistently refractory to explanation with the above two-axis model. Recently, the terpenoid compound methyl farnesoate has been physicochemically demonstrated to exist in circulation at physiological concentrations, in several mecopterid orders, including Diptera. In addition, it has also been recently demonstrated that the receptor to which methyl farnesoate binds with nanomolar affinity (ultraspiracle, an ortholog of retinoid X receptor) requires a functioning ligand binding pocket to sustain the morphogenetic transition to puparium formation. This review evaluates endocrine and pharmacological evidence for developmental pathways reached by methyl farnesoate action, and assesses the participation of the retinoid X receptor ligand pocket in signal transduction to those developmental endpoints. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction The classic model of endocrine orchestration of insect molting and metamorphosis is that it is primarily regulated by the steroid 20-hydroxy ecdysone (20E) and the sesquiterpene JH (Gilbert et al., 2000; Jindra et al., 2013; Fig. 1B here). 20E binds with a nuclear hormone receptor (EcR) that appears to be an insect ortholog of vertebrate FXR or LXR (Bergot et al., 1981; King-Jones and Thummel, 2005). The vertebrate receptor FXR forms a heterodimer with the retinoid X receptor (RXR, Lefebvre et al., 2010) to form a functional complex that is responsive to endogenous FXR ligand. The insect receptor EcR forms a heterodimer with an ortholog of RXR (named ‘‘USP’’ in mecopterid orders that include Diptera), to form the functional ecdysteroid receptor (Henrich et al., 2000). There is evidence that a JH receptor Methoprene-tolerant (MET) may physically bind Abbreviations: RXR, retinoid X receptor; EcR, ecdysone receptor; USP, ultraspiracle; FXR, farnesoid X receptor; LXR, liver X receptor; JH, juvenile hormone; Met, Methoprene tolerant; 20E, 20-hydroxy ecdysone; PTG, prothoracic gland; MDCF, methyl dichlorofarnesoate; MCFs, chlorinated methyl farnesoates; JH, juvenile hormone; bHLH-PAS, basic helix loop helix-per-arnt-sim domain; RA, retinoic acid. ⇑ Corresponding authors. Fax: +1 859 257 1717. E-mail addresses: [email protected] (D. Jones), [email protected] (G. Jones), [email protected] (P.E.A. Teal). 0016-6480/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygcen.2013.09.021

with EcR/USP during ecdysone-driven larval to pupal metamorphosis (Bitra and Palli, 2009; Guo et al., 2012). With the intersection 20E/EcR and JH/MET axes, we have the molecular implementation of the classic endocrine two-hormone model of insect metamorphosis. But is it as simple as this? Is something missing? The classic two-hormone axis of 20E-EcR/JH-MET provides an explanation of the maturation of adult structures of the genetic model Drosophila melanogaster (hereafter Drosophila) (Riddiford, 2012) and other insects (Konopova et al., 2011). However, a body of endocrine and pharmacological data on other developmentally parallel morphogenetic events, such as higher dipteran puparium formation, have not been effectively explained by this classic model (Staal, 1975; Riddiford and Ashburner, 1991; Jindra et al., 2013). Recently, physiological levels of circulating methyl farnesoate have been measured in Drosophila larvae (Jones et al., 2013). In addition, genetic manipulations have established that Drosophila USP, which exerts a nanomolar affinity for methyl farnesoate (Jones et al., 2006), requires a functioning ligand binding pocket in order to support a formation of the puparium at the end of larval development (Jones et al., 2013). In this review, we assess how recent advances in sesquiterpenoid chemistry couple with discoveries in insect nuclear receptor biochemistry and molecular genetics of metamorphosis, to enable a more comprehensive explanation of the array

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Fig. 1. Structures of natural and synthetic terpenoids as potential ligands for vertebrate RXR and mecopterid USP.

of morphogenetic phenomena occurring at the larval to pupal transition.

2. Advances in physicochemical detection of endogenous diterpenoid and sesquiterpenoid ligands Conspicuously absent from many models of the action of vertebrate RXR, and from most models of action of mecopterid USP, is a nanomolar affinity endogenous ligand acting through RXR or USP. Indeed, there has been some consideration as to whether vertebrate or mecopterid RXR/USP even has a nanomolar affinity endogenous terpenoid or other ligand (Wolf, 2006; Markov and Laudet, 2011). However, physicochemical advances in analysis of biological samples have recently enabled the definitive measurement of physiological concentrations of endogenous terpenoids that have nanomolar affinity for binding with RXR or USP. 2.1. Detection of endogenous activating and antagonistic diterpenoid ligands of vertebrate RXR In vertebrates, nanomolar concentrations of the diterpene 9-cis retinoic acid (an RXR activator, Fig. 1G) have recently been reported for the pancreas (Kane, 2012). The results of genetic interference with RXR function (Miyazaki et al., 2010), as well as pharmacological disruption of RXR action (Pérez et al., 2012), are all suggestive of the existence of a vertebrate 9-cis RA/RXR hormone/receptor axis in the pancreas (Kane, 2012). In addition, b-apo-13-carotenone, which has the same nanomolar affinity for RXR as does 9-cis RA, has recently been measured in serum at the same concentration as 9-cis RA (Kane, 2012). However, b-apo-13-carotenone is an RXR antagonist that blocks the action of agonist 9-cis RA at the RXR ligand binding pocket. Hence, no presumption can be made that natural, nanomolar affinity ligands for

RXR (or USP, below) will necessarily be activators (Harrison et al., 2012; Eroglu et al., 2012; Sayin et al., 2013). 2.2. Detection of circulating methyl farnesoate in larval insects Over the years bioassays, radioimmune assays, radiochemistry and various chemical methods and derivatizations have been used to measure juvenile hormone levels, including employment of both HPLC and GC modes of chromatographic separation, and coupled with various detectors for detection of various analytes (Gilbert and Schneiderman, 1960; Pratt and Tobe, 1974; Baehr et al., 1976; Bergot et al., 1981; Mauchamp et al., 1979; Richard et al., 1989; Cusson et al., 1990; Rivera-Perez et al., 2012). Our approach to identification of sesquiterpene esters from hemolymph of insects has been to use gas chromatography employing capillary columns to separate underivatized components of extracts. Coupling of capillary GC columns with mass spectroscopy (GC–MS) allows for identification and quantification methyl farnesoate, and positional and geometric isomers of its epoxidized relatives, the juvenile hormones (Fig. 1; Teal et al., 2000; Jones et al., 2013). By the above method, quantitative determination of methyl farnesoids from blood is possible on single larvae of Drosophila. These advances have enabled the larger and more complex experimental designs to discern the role in Drosophila of various methyl farnesoids in morphogenetic phenomena. 3. Cyclorrhaphous corpora allata secretions and larval development The ring gland of higher (cyclorrhaphous) Diptera is a composite gland, composed of the prothoracic gland, the fused pair of corpora allata and the corpora cardiaca. Many endocrine and biochemical studies have established that the prothoracic gland portion secretes ecdysone, and the corpora allatal cells secrete

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methyl farnesoate and JH (Fig. 1A, B and D). Suppression of biosynthesis of methyl farnesoids results in death primarily at the 2nd– 3rd larval molt of Drosophila, which can be rescued in part with dietary farnesol but not with methyl farnesoate or JH III. This result supports a model in which some combination of methyl farnesoids is necessary to sustain larval development prior to initiation of metamorphosis (D. Jones et al., 2010). In addition, suppression of JH reception appears to prompt certain events associated with the initiation of Drosophila metamorphosis (Abdou et al., 2011). However, the JH field has frustrated itself for many years on the inability to demonstrate JH regulation over certain overt morphogenetic events at higher dipteran metamorphosis, such as puparium formation (Staal, 1975; Riddiford and Ashburner, 1991; Jindra et al., 2013). Hence, pupariation has become popularly modeled as an event ‘triggered by ecdysone,’ without a role for JH. Based on this popular model, genomic studies have sought to discern ecdysone triggered gene expression networks invested in pupariation (Beckstead et al., 2007; Davis and Li, 2013). Yet, a number of endocrine and pharmacological studies reveal that secretion of the corpora allata must be a part of the morphogenetic model of cyclorrhaphan pupariation. 4. Cyclorrhaphous corpora allata secretion can enhance the rate of puparium formation Vogt (1943) published the first data demonstrating that the larval CA of higher dipterans secretes hormones or factors that are active to regulate puparial morphogenesis. She ligated 4 day, 21 h old (4D21h; mid-last) instar Drosophila hydei larvae at the abdomen to prevent its pupariation.* When she implanted a ring gland from 5D21h larvae, 97% of the isolated abdomens pupariated 2–3 days later (none by 1 day later). When she implanted only the left and right excised prothoracic gland (PTG) parts of the gland (the ‘‘schenkelzellen’’ or ‘‘thigh’’ regions), comprising about half of the PTG mass, only 30% pupariated by 3 days later. Implanting four such PTG parts, so as to restore the approximate PTG mass in an intact ring gland, only induced 67% of the abdomens to pupariate by either days 2 or 3. These data suggested that secretion from the corpora allatal part of the ring gland contributed to the effect of the total ring gland implant that caused 97% pupariation. This hypothesis was supported by the intriguing result that implantation of the ‘‘transverse arch’’ portion of this age ring gland (containing the CA and the remaining adjoining portions of the PTG) accelerated the rate of pupariation over even that of an intact ring gland: 10 of 23 pupariating abdomens pupariated by day 1. Importantly, these data indicate that the action of the corpora allata secretions is not indirect through other endocrine or neural organs in the anterior of the animal, but that the puparium forming epidermis itself could be the cellular target. These data also indicated that the outcome of the pupariation event is dependent on a physiological interaction between the CA secretion and the amount of PTG secretion (i.e., pupariation of a ligated 4D21h abdomen was faster when the CA was present with a half a 5D21h PTG equivalent instead of when the CA was present with an entire PTG equivalent). 5. Interplay of CA secretion and ecdysone on puparium morphology The studies of a number of laboratories support the concept that certain stage-incorrect secretions of the CA at the time of a ⁄ Pupariation of D. hydei on average 6 days and 11 h (6D11h) from egg hatch. The ligation ‘head critical period’ for pupariation is ca. 5D21h; wandering starts mid-day 5. The failure of the puparial cuticle to develop proper color was also observed.

pupariation-triggering pulse of ecdysone will disrupt normal puparial morphogenesis. When Hadorn (1937b, D. melanogaster) implanted the mature (‘‘reifen’’) ring glands from late 3rd instar (wandering) larvae into intact early 3rd instar hosts, the recipients developed uncontracted, but tanned puparial cuticle. Each recipient larva thus contained both an endogenous younger ring gland, producing relatively little ecdysone and an exogenous older 3rd instar ring gland, secreting a high amount of ecdysone (Parvy et al., 2005). Hadorn (1937a,b) described the resultant prematurely pupariating ‘‘pseudopupae’’ as exhibiting a tanned cuticle of an otherwise larval (uncontracted) body form. In more closely timed experiments Vogt (1943, D. hydei), implanted various ecdysone-active ring glands into unligated larvae, prior to the 23rd h of the ca. 85 h-long 3rd instar period (approximately similar in timing of host and donor development to that of Hadorn (1937b). Again, the recipients formed sclerotized, tubular-shaped puparia (Fig. 2C vs. D). In addition, the puparial cuticle did not tan the normal full dark red color (notice lighter sclerotized cuticle above and below the marked abdominal segments in Fig. 2E). The failure of the puparial cuticle to develop proper color was also observed when premature pupariation was prompted by implantation of ecdysone-active ring glands into ligated early third instar abdomens. Collectively, these data evidenced, with natural secretions, that a sufficiently premature exposure of early 3rd instar larvae to a pupariation-provoking pulse of ecdysone elicits a deranged puparial process. This interpretation is supported by the results of putting young (<12 h old) 3rd instar Drosophila larvae onto diet containing 20E. Some larvae in response to the pulse of 20E apolysed in less than a day to a pupal body, inside a 3rd instar cuticle that remained larviform and failed to form a puparium (Supplementary Fig. 1). These outcomes suggest that a regulatory event occurring after the early 3rd instar enables larval cuticle to subsequently form a correct puparium in response to a pupariation-provoking pulse of ecdysone. Possompes (Murphy et al., 1993; using Calliphora erythrocephala) came at the question of CA secretion and pupariation from a different direction. Instead of using ligated abdomens, he used recipient larvae from which their own ring gland had been surgically extirpated. He showed that a developmental time point existed during the feeding period of the 3rd instar, at which he could rescue normal puparium formation by implanting the isolated portions of the PTG from the ring gland. However, when the ring gland ‘arch’ (containing a CA and the other portion of the PTG cells) was instead implanted, the recipient larvae failed to contract and fully tan (Fig. 2F vs. G). Hence, at that feeding stage time point, the presence of secretions from a wandering stage CA interfered with the rescue of pupariation that would have otherwise been induced by the portion of the PTG that adjoined the implanted CA. This result was reinforced when Possompes obtained a similar failed puparium formation by implanting isolated adult corpora allata into intact early 3rd instar larval hosts (Fig. 2H). The failed puparium formation arising from incorrect combinations or incorrect timings of CA secretion and ecdysone secretion offers a means to understand the role of CA secretions in regulation of pupariation, i.e., puparium morphology might be signaled (at least in part) by a controlled interaction of CA secretion(s) with ecdysone signaling.

6. Effect on pupariation of genetic manipulation of larval methyl farnesoid production There is some variation among higher dipteran groups as to when normally developing larvae become ‘committed’ that the next occasion of a molt-stimulating pulse of ecdysone will be the occasion of a larval to pupal molt. In Drosophila, it is thought that this commitment occurs toward the end of the 2nd instar (Jones

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Fig. 2. Larviform puparia arising from endocrine missignaling. Shown for the indicated species of cyclorrhaphan Diptera are normal puparia (A, C and F), and tubular, larviform puparia arising due to the indicated treatment (in blue) of last instar larvae by either implantation of ring glands (D, E and G), implantation of adult corpora allata (H), or topical treatment with a mixed function oxidase inhibitor (B) that may slow catabolism of endogenous methyl farnesoids (Staal [57]). The red line alongside panel D denotes a darker area of the cuticle in contrast to surrounding lighter areas. (Panel I) Diagrammatic representation of pupariation outcomes of treatment of 3rd instar Drosophila larvae with dietary 20E or implantation of ring glands actively secreting ecdysone. Generalized curves of methyl farnesoate and JHIII titer in hemolymph are abstracted from Jones et al. (2013) and Fig. 3 here. Images in upper figure adapted from the following sources: B – Wright (1970); C, D, E – Vogt (1943) and F, G, H, – Possompes (1953).

and Jones, 2007). In calypterate flies there is lability in this commitment into the early feeding stages of the final larval instar (Zdárek and Sláma, 1972). Collectively, the implantation/extirpation studies offer the hypothesis that subsequent to the above commitment during the late 2nd instar there is during the feeding stage of the 3rd instar a

regulatory event of interaction of CA secretion with ecdysone signaling. However, the above studies did not chemically identify the operative secretion. Recently, using the enhanced physicochemistry described in Section 2, we showed that a large peak (near 500 nM) of circulating methyl farnesoate occurs during the late feeding stage of the 3rd instar (Jones et al., 2013; Fig. 3 here). This

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A B

C

D

Fig. 3. Larviform puparia arising from reduced methyl farnesoid titers. (A) Physicochemical measurements of the circulating methyl farnesoids in normal D. melanogaster larvae compared to late feeding stage larvae expressing broad-RNAi (BDSC 27272) or usp-RNAi (BDSC 27258) in the corpora allata of the ring gland using a Di11 driver. Measurements are either adapted from those reported by Jones et al. (2013; last three groups) or performed with the methods of Jones et al. (2013) using larvae under similar rearing conditions (first three groups). In larvae expressing either RNAi, methyl farnesoate was the most strongly reduced in concentration (<1% normal), compared to JH III (26–125% of normal) or bisepoxyJHIII (4–25% of normal). The larvae expressing broad-RNAi and usp-RNAi (C and D) subsequently failed to form a normal puparium, instead very weakly sclerotizing the cuticle while remaining larviform in shape.

result offers a more specific hypothesis: this mid-late 3rd instar feeding stage peak in methyl farnesoate is involved in a regulatory event that signals or enables the proper formation of a puparium. In preliminary studies, we used Drosophila molecular genetic approaches to address this hypothesis from a different direction: altering the endogenous level of circulating methyl farnesoids through misfunction (but not ablation) of the intact larval corpora allata. We considered that if (only) the larval corpora allata cells were unable to respond to larval stage developmental signaling by circulating ecdysteroids, then those ‘confused’ CA cells may not mature to produce the large late feeding stage peak in methyl farnesoate. Toward that end, we used the Di11 corpora allata-specific driver (Belgacem and Martin, 2007; Mattila et al., 2009) to drive expression in the corpora allata of RNAi against broad, which is considered as a regulator necessary for larval to pupal maturation (Karim et al., 1993; Zhou and Riddiford, 2002; Zhou et al., 2004). The intriguing outcome was that all larvae with suppressed broad in the corpora allata cells exhibited failure to form a proper puparium. After a period of wandering behavior, the larvae stopped crawling and strongly reared up the head and thorax (Fig. 3D). Over the next several hours the larvae only weakly formed features of a puparium on the head and thorax (partial eversion of anterior spiracles, withdrawal of mouthparts, weak deformation of larval head cuticle), and then totally stopped all movement, with no further progress on puparium development (Fig. 3D). The body remained tubular or larviform, the cuticle remained soft or very faintly sclerotized but did not tan, and the animals desiccated over the next day. Ecdysone signaling during the 3rd instar is also considered to be a necessary signal for maturation of the corpora allata. We considered that targeted disruption of 20E reception in the corpora allata cells could also ‘confuse’ the maturation of these cells. Larvae with Di11-driven suppression of usp (a necessary heterodimer partner of EcR for ecdysone-signaling) showed a phenotype behaviorally and morphologically nearly

identical with that for suppression of broad (Fig. 3C). The control treatment of Di11-driven suppression of Methoprene-tolerant (Met) exhibited no effect and all larvae formed normal puparia and emerged as adults (not shown). Hence, in both cases that we specifically sought and achieved strongly reduced methyl farnesoid titer during the late feeding stage the puparium failed to form. It has been postulated that a small increase in ecdysone production that occurs during the late feeding stage is responsible for triggering the end of larval feeding and commencement of the developmental pathway toward puparium formation (Riddiford, 1993; Parvy et al., 2005). The classical endocrine transplantation data (Sections 3–5, Fig. 2, above) evidenced that both corpora allata secretion and ecdysone, in proper balance, are necessary cosignals for puparium formation, but that excess ecdysone at that signaling can derange puparium formation. The more recent data summarized above, using measurements and effects of endogenous secretions of the corpora allata themselves, support a more specific hypothesis that a mid-late feeding stage increase in methyl farnesoate is also necessary, in addition to 20E, for puparium formation. Hence, a hypothesis that calls for involvement of a hormone/receptor axis in addition to 20E/EcR provides a more effective framework to account for orphaned reports of the disruption of puparium formation by exogenous methyl farnesoate-type compounds.

7. Evidence that JH III is not a regulator of puparial morphogenesis 7.1. Larval treatment with JH or JH analog does not disrupt puparium formation The above studies utilized or examined endogenous secretions of the corpora allata, the most studied of which has been JH III. It

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Drosophila melanogaster Normal JH IIIl Methoprene

D

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Normal

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Musca domesca Methyl MDCF Farnesoate

MCFs

Fig. 4. Puparial phenotype of dipteran larvae feeding on diet treated with sesquiterpene-like molecules. Panels A–C, Drosophila melanogaster; D–G, Musca domestica. In B and C, larvae were reared under the conditions of Harshman et al. (2010), 2.5  10 5 moles of JH III or methoprene/4.9 cm2 surface area). House fly larvae in Panels E–H were reared on 2 ml of diet (4.9 cm2 surface area) onto which was dispensed methyl dichlorofarnesoate (MDCF, Supplementary Fig. 2A), or a mix of chlorinated methyl farnesoates (MCFs, Supplementary Fig. 2B) or methyl farnesoate (MDCF, MF at 1 part per 10,000; MCFs 1:30,000, by weight:volume) (n > 25 for each group). As shown, some larvae treated with these compounds externally sclerotized and tanned the cuticle on their tergal or sternal plates, but with a very larviform puparium, before dying without apolysis, a result that was not seen in carrier controls or those exposed under the same rearing conditions to 1:10,000 methoprene (n = 20).

has recently been established that a properly timed lowering of the JH titer during later larval development of Drosophila contributes to coordination of the metamorphic transformation of larval fat body (Abdou et al., 2011; Liu et al., 2009). If the properly timed lowering of the JH titer is also a necessary signal for Drosophila puparium formation, then exogenous treatment with JH or JH analogs would be predicted to interfere with puparium formation. However, even high doses of dietary JH III did not prevent most Drosophila larvae from attaining pupariation, although the treatment strongly suppressed the resultant pupae from subsequently developing to the adult (Harshman et al., 2010). The puparium attained by larvae exposed to high concentrations of JH III in the diet is of substantially normal morphology (Wilson and Fabian, 1986, Fig. 4B here). In additional reports, D. melanogaster larvae were instead reared on food containing synthetic structures that were commercially designed to highly maximize ‘JH activity’ to disrupt formation of imaginal structures. A concentration of 1:10,000 (pyriproxifen) or even up to 1:1000 (methoprene) was highly active to disrupt imaginal morphogenesis, yet only ‘‘delayed, but failed to prevent, pupariation of most larvae,’’ (Riddiford and Ashburner, 1991). Again, the

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Wild Type

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Blocked JH Reception

puparia resulting from feeding with these compounds were of substantially normal morphology (Riddiford and Ashburner, 1991; Riddiford et al., 2010; Fig. 4C here), while the formation of adult structures by the pupae inside was strongly suppressed. These data for exogenous treatment with JH III and JH analogs do not suggest that a lowering of the JH titer during the later larval feeding stage is a necessary signal for Drosophila puparium formation. 7.2. Ablation of JH receptors does not disrupt puparium formation The above pharmacological evidence that the JH titer during the later larval feeding stage is not a necessary signal for puparium formation is further supported by studies that instead genetically ablated JH reception. It has been proposed for Drosophila that both GCE and MET are receptors that transduce developmental actions of JH (Liu et al., 2009; Riddiford et al., 2010; Bernardo and Dubrovsky, 2012). When larvae were made null for both the gce and Met, the third instar larvae developed successfully to attain an essentially normal puparium, then next apolysed to the pupal body, and finally died shortly after pupal head eversion (Abdou et al., 2011; Fig. 5A–C here). No tubular or larviform puparia were

D

E

Blocked Reception Through USP

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G

Rescued Reception Through USP

Fig. 5. Loss of receptors for JH III does not block puparium formation. (Panel A) Wild type puparium (note puparial operculum encircled by white dots); puparial ‘horns’ (larval anterior spiracles) denoted by black arrow. (Panels B and C) Animals that are null for both Met and gce form a puparium, before arresting development shortly after the attempted eversion of the pupal head. (Panels D and E) Animals null for endogenous usp and expressing only a transgenic mutant USP with much reduced affinity for methyl farnesoate do not form a puparium, even though they apolyse to the pupa (images adapted from Jones et al., 2013) (Both panels are USP mutated to alanine at position N325). (Panels F and G) Null usp2 animals rescued to adult form by a transgenic HA-tagged wild type Drosophila USP (F) or wild type Aedes aegypti USP isoform A (G), each expressed under the control of the natural Drosophila USP promoter. Photos in B and C were kindly provided by Dr. Jian Wang.

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reported. These results strongly argue that methyl farnesoid signal transduction through either GCE or MET is not necessary for either attainment of pupariation or normal puparium morphology. 8. Evidence that methyl farnesoate contributes to puparial morphogenesis The calypterate Diptera also appear more sensitive than is Drosophila to disruption in cuticular pupariation by exogenous methyl farnesoate structures. Dietary methyl farnesoate, that would add to the high endogenous methyl farnesoate peak during the late feeding stage (Fig. 3A), did not cause abnormal morphology of Drosophila puparia (D. Jones et al., 2010); Supplementary 1A), which is not an unexpected result. However, we have observed a low incidence of larval-puparium intermediates in (calypterate) house flies reared on dietary methyl farnesoate (Fig. 4F; Supplementary 1B). More consistent effects on calypterate dipteran puparia have been reported in response to treated with halogenated analogs of methyl farnesoate, that may be more metabolically stable and/or have some misfit to and hence render some misfunction to the cognate methyl farnesoate target. Morgan and LaBrecque (1971) reported that topical treatment of late last instar house flies with an undefined mixture of chlorinated ethyl farnesoates caused the majority of larvae to stop development not later than shortly after puparium formation (prior to the air bubble/water buoyant stage that occurs just after apolysis to the pupa; Bainbridge and Bownes, 1981). Subsequent studies more closely focused on the action of individual chlorinated methyl farneosate compounds. When house fly eggs were treated with methy7,11-dichlorofarnesoate, ‘‘a considerably high proportion of the larvae’’ formed elongated, worm-like puparia (i.e., tubular in shape) (Matolin, 1971; Supplementary 1C here). We recently tested a freshly made stock of a 96% pure stock methyl-7,11-dichlorofarnesoate (MDCF, Echelon; Supplementary Fig. 2), by placing 1st instar house fly larvae on artificial diet that was surface-treated with the total equivalent of a 1:10,000 ratio of compound:food (weight/volume). In preliminary observations we confirmed the above report that some larvae formed larviform puparia (Fig. 4E), which died shortly after the abnormal pupariation (at or before air bubble formation). Other chlorinated methyl farnesoates also caused abnormal pupariation (MCFs, Fig. 4F); feeding exposure beginning at the 3rd instar was sufficient to cause tubular puparia. Of the puparia that were not larviform following MDCF or MCF treatment, most produced emerging adult flies. In contrast, we did not observe such larviform puparia for animals reared under the same conditions on media treated instead with 1:10,000 methoprene. However, methoprene treatment was much more effective to prevent subsequent emergence of adult flies than was MDCF (as was reported for topical treatment; Sehnal and Zdarek, 1976). Relatedly, topical treatment of 3rd instar house flies with the 2,3-methylene derivative of ethyl farnesoate (Fig. 1E) was more active to prevent attainment of puparium formation than treatment with the 11-methoxy ‘methoprene-like’ variant of this compound (Fig. 1F). Reciprocally, the methoprene-like variant compound was more active than the former in preventing pupariated animals from then attaining adult eclosion (Styczyn´ska et al., 1976). 9. Weakened USP binding to ligand disrupts larval pupariation The hypothesis that binding of endogenous ligand by USP is necessary for USP function in vivo was recently tested by point mutating the ligand binding pocket of USP so as to significantly reduce its affinity for methyl farnesoate; the mutant USP was then challenged to sustain normal larval development. When Drosophila

larvae were made null for ultraspiracle (a lethal condition for death at the first larval molt; Perrimon et al., 1985; Henrich et al., 2000), a transgenic rescue with a wild type USP (under the control of the natural ultraspiracle promoter) enabled survival of the larvae to pupariation and onto morphogenesis of the adult form (Henrich et al., 2009, Fig. 5F and G here). When instead the rescue transgene encoded a USP that was point-mutated for reduced affinity for methyl farnesoate, the larvae developed through the ecdysone-driven first two larval molts. However, none of the 3rd instar larvae formed a normal puparium (Jones et al., 2013; Fig. 5D, E here). Cultured cell transfection studies confirmed that the ligand pocket mutation did not in and of itself physically impart an allosteric effect to cause the EcR partner to fail to respond to its ligand, 20E (Jones et al., 2013). Most larvae with the strongest point mutations to the ligand binding pocket wandered and became sessile with a larviform body, and then apolysed to cryptocephalic pupae, omitting the step of puparium formation. The cryptocephalic pupae proceeded to then express a pupal-specific cuticle gene, showing that the developmental lesion was not a general developmental block prior to puparium formation, but rather a specific failure of puparium formation (Jones et al., 2013).

10. Summary and prospects 10.1. Methyl farnesoate and USP as necessary signaling components for pupariation The data reviewed here on model cyclorrhaphous flies demonstrate the sensitivity of puparium formation to disruption of methyl farnesoid signaling. The classical gland transplantation studies of Hadorn, Vogt and Possompes showed that undersignaling, oversignaling or mistimed signaling from larval corpora allata, in relation to 20E signaling, deranges the morphogenetic events of puparium shape, color and/or sclerotization (Fig. 2A). Those studies also indicated that the hormonal factor from the corpora allata acts during the 3rd instar, prior to the wandering stage (Fig. 2B). Those authors considered whether the acting hormone might be the same as the juvenile hormone that is necessary for the larval–pupal transition, but the molecular genetic and physicochemical tools did not exist that that time to further discern its chemical identity. However, recent molecular genetics has demonstrated that total loss of Met and gce expression (the two generally acknowledged receptors for JH III) does not prevent larvae from formation of essentially normal puparia. Another secretion of the corpora allata, methyl farnesoate, was recently physicochemically shown to circulate during the 3rd instar at a much higher concentration than does JH III. The timing of the last instar peak of methyl farnesoate approximates the timing of a small peak in ecdysone that prompts cessation of feeding and puparium formation. Treatment of early 3rd instar Drosophila larvae with a pulse of ecdysone prior to a critical point during the mid-late feeding stage also results in apolysis to the pupal body but without normal puparium formation. Genetic loss of the nuclear hormone receptor with nanomolar affinity for methyl farnesoate (USP) results in failed formation, coloration and sclerotization of a puparium. In comparison to most JH III analogs, exogenous methyl farnesoate or halogenated methyl farnesoate analogs are more active to prevent formation of a puparium or to cause misformed puparia than are these compounds to cause defects in morphogenesis of adult structures. Collectively, the leads from the classical endocrine studies, and the recent biochemical, and molecular genetic data, together evoke a model in which the hormone methyl farnesoate and the receptor ultraspiracle intersect with ecdysone/EcR signaling during the late feeding stage, and are necessary for normal puparium formation. This model is very different from either a ‘pupariation is controlled

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by ecdysone’ model or the classic JH/ecdysone model of metamorphosis. 10.2. Is methyl farnesoate signaling the sufficient farnesoid signal? Is the above expanded model itself yet sufficient? A limitation is that an experimental design has not yet been identified under which exogenous methyl farnesoate rescues deficiency in endogenous methyl farnesoate signaling. A clue as to why may be offered by the recently published methyl farnesoid titers during the final instar: a peak in the circulating concentration of bisepoxyJH III occurs at a similar stage as the peak in methyl farnesoate (Fig. 3). That is, it could be that methyl farnesoate is a necessary, but not sufficient, methyl farnesoid signal, and that bisepoxyJHIII signaling is also necessary. The functional larval roles in vivo of bisepoxyJHIII remain the least understood of the three primary sesquiterpene secretion products of the corpora allata. Thus far, the published methods that have been apparently successful to lower endogenous production of methyl farnesoate are nonselective. That is, apoptotic damage of the corpora allata (Liu et al., 2009; Riddiford et al., 2010), suppression of methyl farnesoid biosynthesis by HMRCR-RNAi (G. Jones et al., 2010), and ‘confusing’ the corpora allata as to their development stage (Fig. 3 here) all affect, to some extent, production of all three methyl farnesoids. In the case of HMGCR-RNAi, provision of dietary farnesol, which would be hypothesized to enable resumed production of all methyl farnesoids, did provide rescue to puparium formation, while methyl farnesoate alone, JH III alone and these two hormones in combination did not provide rescue (G. Jones et al., 2010). In the case of developmentally ‘confused’ corpora allata, dietary farnesol would not necessarily be predicted to cause rescue, and we have been unable to induce rescue of puparium formation with either dietary methyl farnesoate or JH analog (methoprene). As discussed elsewhere (Jones et al., 2013), on kinetic grounds we believe it unlikely that USP is a receptor for signaling by bisepoxyJHIII. Future detailed studies to test a potentially necessary role of bisepoxyJHIII in puparium formation are sorely needed, as well as identification of the endogenous receptor for this hormone as being MET, GCE, or some other molecule. 10.3. Mechanism of USP transduction of signaling At present, the mechanism by which ligand binding is physically transduced by USP into regulatory signaling is unknown but several possibilities, reviewed below, have been indicated that are not mutually exclusive and the exercise of which may depend on cell type, promoter context, and dimer partner, as discussed below and outlined in Fig. 6. 10.3.1. Physical competence to bind ligand The full-length Drosophila ortholog, USP, when purified under standard conditions and concentration, exhibits a nanomolar affinity for methyl farnesoate (Fig 6A; Jones et al., 2006), similar in affinity to that of human RXR binding to either 9-cis RA (a natural RXR agonist) or b-13-apocarotenone (a natural RXR antagonist) (Kane, 2012). This binding can be competitively inhibited by the synthetic RXR ligand tributyltin (Jones et al., 2013) that forms a near covalent bond with a ligand pocket cysteine residue in crustacean RXR that is conserved in Drosophila USP (le Maire et al., 2009). JHIII exhibits a weaker (micromolar) affinity (smaller arrow, Fig. 6A), but at sufficient concentration will saturate the USP binding site. While USPs overexpressed and superconcentrated under certain conditions for crystal formation tend to become fortuitously occluded with phospholipid from the local expression system (Hill et al., 2013), Clayton et al. (2001) succeeded to obtain an estimated 25% of even the crystallized Drosophila USP to possess a vacant ligand binding pocket,

Fig. 6. Summary of ligand binding, conformational change and transcriptional modulation capabilities of Drosophila USP. Diagrammed are dynamic changes in homodimerization and heterodimerization state of USP and EcR, as influenced by potential farnesoid-like or nonfarnesoid ligands of USP, and as influenced by the DNA binding site and its promoter context. Transcriptional activity of reporter genes conferred by the shown dimers in the various liganded states are indicated by size (relative activity) and color (green, increased activity; red, decreased activity) of the arrows. In vivo, all of the indicated responses to ligand could vary in the context of cell type. MF = methyl farnesoate; JH = juvenile hormone; TBT tributyltin. Diagram based primarily on references Crossland and Geoff (2002), Fang et al. (2005), Henrich et al. (2000), Jones and Jones (2007), D. Jones et al. (2010), G. Jones et al. (2010), Maki et al. (2004) and Xu et al. (2002). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

available to receive ligand. Sasorith et al. (2002) showed by computational methods that a vacant ligand pocket in Drosophila USP of similar general size and shape as vertebrate RXR would be able to accommodate the methyl farnesoid structure. 10.3.2. Structural response to ligand Independent laboratories have demonstrated that Drosophila USP is structurally competent to respond to ligand binding by changing its quaternary structure (Fig. 6). Purified full length Drosophila USP exists in solution as primarily monomer but the minor homodimer form can be captured by dimethyl suberimidate crosslinking (Jones et al., 2001). When JHIII was used as a probe at a concentration that is saturating relative to its Kd, (Jones et al., 2001) it stabilized the homodimer as indicated anisotropy measurements (Fig. 6B). In a plant cell reporter system (Crossland and Geoff, 1996), the more farnesoid-like JH analog methoprene (Fig. 1H) strongly induced USP ligand binding domains to homodimerize, but disrupted the formation of the EcR/USP heterodimer, whereas the nonfarnesoid-like JH analog fenoxycarb was much less effective in either situation (Fig. 6D). As noted by Lezzi et al. (2002), the presence and appropriate configuration of USP binding motifs in DNA (green underline, Fig. 6B) further promotes stability of the full length USP homodimer. 10.3.3. Functional performance in response to ligand Several laboratories have shown that USP has a functional ability to become transcriptionally activated by ligand if the appropriate DNA binding sites are present and if the appropriately crafted core promoter is used for the cell type in the assay. Using JHIII as a probe at saturating concentration, DR12 as a binding site, and a

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JH-sensitive core promoter as a reporter, Xu et al. (2002) showed by mutational analysis that the ligand binding pocket of full length USP transduced transcriptional activation of the reporter. In experiments that manipulated the intracellular (full length) USP or EcR levels, and used instead a DR1 binding site (Fang et al., 2005), the intracellular conditions that favored USP homodimer enabled transcriptional activation of the reporter by JHIII alone, while conditions that favored EcR/USP heterodimer prevented activation by JHIII alone (Fig. 6C) but enabled JHIII to enhance activation by 20E (Fig. 6D). In a COS cell system (Henrich et al., 2009), saturating concentration of several farnesoids, including methyl farnesoate, did not alone activate Drosophila USP but did potentiate 20E activation of the EcR/USP heterodimer (Fig. 6E). Relatedly, the RXR activator tributyltin can activate crustacean RXR when only RXR is present; in the presence of both EcR and RXR, TBT cannot activate RXR but can potentiate 20E activation of EcR (Wang et al., 2011). This same TBT ligand can modulate ecdysone signaling in vivo in the dipteran Chironomus (Morales et al., 2013). In a different cell culture and reporter system (Maki et al., 2004), JHIII acting through USP instead partially suppressed 20E activation (Fig. 6F). It is apparent that dipteran USP is competent to bind ligand, be conformationally changed thereby and exert transcriptional modulation. However, the nature of the tertiary or quaternary conformational change, or transcriptional modulation, is highly dependent upon cell type, DNA binding site, promoter context and the nature of the dimer partner. At this time there is insufficient information to postulate a particular mechanistic model by which methyl farnesoate signaling is transduced through USP for specifically the regulation of pupariation, because no primary target gene for that morphogenetic effect of methyl farnesoate has yet been identified. Next generation molecular approaches, such as ChIP-seq, enable genome-wide identification of all changes in nuclear binding sites of a hormone receptor under conditions of normal and altered signaling. Such an approach, together with genome-wide expression analysis, will enable identification of genes for expression changes and at which USP binds (or changes in binding), when normal methyl farnesoate signaling is altered. These genes would then provide leads on potential direct targets of methyl farnesoate signaling to regulate morphogenetic events of pupariation. Acknowledgments G.J. and D.J. were supported by NSF 1052142. Appreciation is expressed to Drs. Lynn Riddiford and Judy Willis for their very helpful reviews of an early version of the manuscript, and to the two anonymous reviewers whose suggestions rendered further improvement.

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