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Proc Natl Acad Sci U S A. Jul 15, 2008; 105(28): 9540–9545.
Published online Jul 7, 2008. doi:  10.1073/pnas.0712132105
PMCID: PMC2474474
Biochemistry

Locust retinoid X receptors: 9-Cis-retinoic acid in embryos from a primitive insect

Abstract

The retinoid X receptor (RXR) is activated by its often elusive cognate ligand, 9-cis-retinoic acid (9-cis-RA). In flies and moths, molting is mediated by a heterodimer ecdysone receptor consisting of the ecdysone monomer (EcR) and an RXR homolog, ultraspiracle (USP); the latter is believed to have diverged from its RXR origin. In the more primitive insect, Locusta migratoria (Lm), RXR is more similar to human RXRs than to USPs. LmRXR was detected in early embryos when EcR transcripts were absent, suggesting another role apart from ecdysone signaling. Recombinant LmRXRs bound 9-cis-RA and all-trans-RA with high affinity (IC50 = 61.2–107.7 nM; Kd = 3 nM), similar to human RXR. To determine whether specific binding had functional significance, the presence of endogenous retinoids was assessed. Embryos were extracted by using modified Bligh and Dyer and solid-phase protocols to avoid the oily precipitate that makes this material unsuitable for assay. These extracts contained retinoids (5.4 nM) as assessed by RA-inducible Cyp26A1-promoter luciferase reporter cell lines. Furthermore, the use of HPLC and MS confirmed the presence of retinoids and identified in any embryo, 9-cis-RA, in addition to all-trans-RA. We estimate that whole embryos contain 3 nM RA, including 9-cis-RA at a concentration of 1.6 nM. These findings strongly argue for a functional role for retinoids in primitive insects and favor a model where signaling through the binding of 9-cis-RA to its RXR is established relatively early in evolution and embryonic development.

Keywords: all-trans-retinoic acid, Locusta migratoria, ultraspiracle

Insect development and metamorphosis are directed by two principal lipophilic hormones: 20-hydroxyecdysone (20-OH-Ec), the active molting hormone, and juvenile hormone (JH), whose titer determines the nature of the molt (1, 2). As demonstrated in the fruitfly, Drosophila melanogaster (Dm), 20-OH-Ec binds to the ecdysone receptor (EcR), which in turn is bound to its obligate heterodimerization partner ultraspiracle (USP), a homologue of the vertebrate retinoid X receptor (RXR) (36). As members of the nuclear receptor superfamily, EcR and USP/RXR share a common modular structure (7) comprised of a N-terminal variable domain (A/B), a DNA binding domain (C), hinge (D), and C-terminal ligand-binding domain (LBD; or domain E/F).

The vertebrate RXRs are known heterodimeric partners of several members of the nuclear receptor superfamily, including the retinoid, thyroid, and vitamin D receptors (8). As demonstrated in vivo, these RXRs can also form homodimers and conceivably mediate an independent retinoid signaling pathway (9, 10). Indeed, the vertebrate RXRs are known ligand-activated transcription factors that bind 9-cis-retinoic acid (9-cis-RA), a stereoisomer of the vitamin A derivative, all-trans-RA (11, 12). RA receptors (RARs), reported only in vertebrates, are distinct in that they bind both all-trans-RA and 9-cis-RA with high affinity (13, 6). In contrast to the vertebrate RXRs, crystal structures reveal that DmUSP and the USPs from the moth Heliothis virescens and the flour beetle Tribolium castaneum probably adopt an inactive conformation (1416) and are unlikely to have an activating ligand. Nevertheless, Jones et al. (17) have suggested that USP could be a JH receptor because it binds methyl farnesoate, an unepoxidated derivative of JH III (Kd = 44 nM) and JH to a lesser extent (Kd = 6700 nM). A high-affinity JH receptor, Methoprene-tolerant (Met; Kd = 5.3 nM) has been recently identified in Dm (18).

Remarkably, the LBD of the protein corresponding to USP from the primitive insect, Locusta migratoria (Lm), shows greater identity to the vertebrate RXR than to USPs of more advanced insects (19). In silico modeling of the LmRXR-L ligand binding pocket also emphasizes amino acid and tertiary structural similarity to the human RXRγ (hRXRγ) (19). In Lm and the German cockroach, Blattella germanica, RXR transcripts have been detected during early embryonic development, even before the appearance of EcR transcripts (20, 21). Thus at least in these two insects it is possible that this EcR binding partner could have a second function. To explore this possibility, two RXR cDNAs were isolated from Locusta. Long and short isoforms, LmRXR-L (GenBank accession no. AY348873) and LmRXR-S (GenBank accession no. AF136372), respectively, differ only by the presence/absence of 22 aa in their LBDs.

Results

Recombinant LmRXR-S was expressed by using the phage T5 promoter in pQE-32 after transfer to Escherichia coli M15[pREP4] cells. Under similar conditions, LmRXR-L expression was disappointing (results not shown). Therefore the hinge and ligand binding domains of LmRXR-L were subcloned into pET-15b [LmRXR-L(DE)] and expressed in BL21(DE3) cells by using the T7lac promoter. As a control for this shorter (≈28 kDa) sequence, the ≈29-kDa human-derived sequence, hRXRα(DE), was similarly expressed. Typically, the purification protocol (a total of 10 purifications were done for the long and short isoforms) yielded 0.8 mg/ml LmRXR-S and 1.7 mg/ml LmRXR-L(DE) protein [see supporting information (SI) Fig. S1 A and B; lanes 7 and 8). Expression and purification of hRXRα(DE) normally yielded 1.0 mg/ml. These preparations were used for assays and antibody production.

Polyclonal antibodies made against purified hRXRα(DE) and LmRXR-L(DE) were used for immunological detection by Western blotting (see Fig. S1 CE). The high similarity between the expressed human and locust RXR LBDs is demonstrated by the observation that the hRXRα(DE) antibody cross-reacted with purified LmRXR-L(DE) and the LmRXR-L(DE) antibody cross-reacted with the purified hRXRα(DE). Western blots further demonstrated the presence of cross-reacting material to the anti-LmRXR-L(DE) in whole embryo extracts (see Fig. S1E).

Ligand Binding Assays.

Although control insect protein did not bind to [3H]-9-cis-RA (data not shown), when recombinant LmRXR-S, LmRXR-L(DE), and hRXRα(DE) were incubated with [3H]-9-cis-RA, there was evidence of binding (Fig. 1). Competition with unlabeled 9-cis-RA showed that for all three protein preparations, binding was specific and very similar. The IC50 for 9-cis-RA by hRXRα(DE) was 74.2 nM, in reasonable agreement with a previously published value (5.6 nM) (22). LmRXR-S showed 1.8 times the affinity (IC50 = 61.2 nM) for 9-cis-RA than did LmRXR-L(DE) (Table 1). When the unlabeled stereoisomer all-trans-RA was used to displace [3H]-9-cis-RA, LmRXR-S again showed slightly more affinity (IC50 = 75.0 nM) than LmRXR-L(DE) for the retinoid, but both retinoids had similar overall competition curves (Fig. 1 A and B and Table 1). Scatchard analysis resulted in a calculated Kd for LmRXR-L(DE) of 3.0 nM (Fig. 1).

Fig. 1.
Retinoid binding analysis. Competitive binding of 9-cis-RA (A), all-trans-RA (B), DHA (C), and methoprene acid (D) with [3H]-9-cis-RA to the purified receptors LmRXR-Short ([filled triangle]), LmRXR-L(DE) (●), and hRXRα(DE) (■). (A Inset ...
Table 1.
Competitive binding data

Competition binding assays with other potential ligands showed lower affinity for the proteins. The affinities of the polyunsaturated fatty acid, docosahexaenoic acid (DHA), and the JH analogue, methoprene acid, were two and three orders of magnitude lower, respectively, than 9-cis-RA for LmRXR-S (Fig. 1 C and D and Table 1). The two nonretinoids were also not as effective competitors for 9-cis-RA binding to LmRXR-L(DE). Overall, the IC50 for DHA was similar for both RXR isoforms but the IC50 of methoprene acid for LmRXR-S was 30% that of LmRXR-L(DE).

Retinoids in Locust Embryos.

The analysis of early locust embryos was difficult because of the presence of yolk and a tough chorion or egg shell. A procedure was developed in several initial trials by “spiking” extracts with 500 ng of 9-cis-RA and radio-labeled [3H]-9-cis-RA (500:0.15 ng). Many extraction methods using ethyl acetate and hexane and numerous solid-phase extractions were also tried. Finally, a modified Bligh and Dyer (23) procedure combined with tandem solid-phase extraction was selected because it resulted in 42.6 ± 9.9% (n = 3) recovery of the added [3H]-9-cis-RA label (data not shown). Embryos were extracted twice to optimize retinoid recovery and avoid the problem of precipitation of an embryo-derived brown viscous oil during evaporation.

Retinoid reporter cells were used to detect the presence of endogenous retinoids in Locusta embryo homogenates and extract preparations (24). These cells proved sensitive to very low concentrations (0.1 nM) of all-trans-RA and all-trans-RA/9-cis-RA suspensions, resulting in luciferase reporter activity that was 2.5- and 2.2-fold greater than DMSO control treatments (Fig. 2). Dilutions of locust embryo homogenates (1:10 dilution) did not induce a significant reporter response. However, preparations of embryo extracts (see Experimental Procedures) resulted in significantly greater reporter activity (2.5-fold) than DMSO controls (P < 0.05). Concentration determination using standard curves generated by a range of 9-cis-RA and all-trans-RA diluted in 100% DMSO indicated the presence of retinoids in total embryonic tissue at a concentration of 1.6 ng/g (n = 4) or ≈5.4 nM.

Fig. 2.
Response of retinoid reporter cell lines to locust embryo extracts. Mouse P19 cells were incubated with locust embryo homogenates or extracts. Retinoids were suspended in 100% DMSO, and this solvent was used as a control, along with all-trans-RA, and ...

Because in vitro experiments indicated the presence of retinoids in Locusta embryos, HPLC-MS was used to determine the presence of specific RAs in embryo extract. Monitoring the eluant from HPLC at 350 nm did not resolve a significant retinoid peak relative to control protein samples (Fig. 3Upper). However, single reaction monitoring and multiple reaction monitoring (MRM) of the transition from 299 to 255 in chromatograms of the Locusta extract preparations showed a significant peak with retention time of 32.8 min with a shoulder at 33.4 min. The peak components exactly comigrated with standards for 9-cis-RA and all-trans-RA, respectively (Fig. 3 Lower). These experiments were repeated several times with isomer standards. The locust extract (3 g) preparation yielded 1.17 ± 0.27 ng (n = 3) of total retinoid. Diluted locust extracts (1.5 g BSA) yielded similar peaks and retention times (32.8 and 33.3 min, respectively) but approximately half the mass (0.64 ± 0.17 ng; n = 3; data not shown). Retinoid peaks were not seen when no embryo extracts or 3 g of BSA were chromatographed. Furthermore, blank runs of samples involving a single isomer did not result in the isomerization of one isomer into the other, suggesting that 9-cis-RA was not a methodological artifact. Thus both 9-cis-RA and all-trans-RA were present in embryos. Integration of the area under the curves suggested that the majority (54.6%) of the retinoid was 9-cis-RA. Using this method and based on total retinoid mass and embryo volume, a calculated 1.3-nM retinoid was detected. Analysis of radiolabeled 9-cis-RA in extracted samples indicated a recovery of 43% of 9-cis-RA; accordingly, there would be ≈3 nM of identified total retinoid, including 1.6 nM of 9-cis-RA in whole Locusta embryos.

Fig. 3.
HPLC-MS analysis of locust embryo extracts. A typical chromatograph of control protein samples (BSA; broken lines) and day-5 Locusta embryo extract preparations (solid lines). (Upper) Elution profile of the HPLC, monitored at 350 nm, with the scale shown. ...

Discussion

Locust RXR Characterization.

Recently, we described the cloning and characterization of two locust RXR isoforms (20) and noted that the LBDs showed greater identity to the vertebrate RXR LBDs than to the USP receptors from moths and flies. Furthermore, RXR transcripts appeared to be present throughout embryogenesis, despite the absence of message for EcR until midembryogenesis (20). This finding appeared curious because EcR is RXR's supposed obligate heterodimerization partner in insects. Similarly, in the cockroach, RXR-L transcripts are detected at the beginning of embryogenesis, before the initial ecdysteroid pulse and RXR-S message appears later in midembryogenesis (21). Together, these observations suggested that RXR in locusts and possibly other primitive insects and arthropods might have an additional role during early development, similar to the function of vertebrate RXR, and in contrast to the orthologous USP of more advanced insect species.

Recombinant LmRXR-L(DE) and hRXRα(DE) were expressed and used to generate antibodies. Immunochemical assays showed that the sequence similarity of the locust and human proteins was also reflected in epitope determinants because there was strong antigen–antibody cross-reactivity with antisera derived from either insect or vertebrate proteins. These antibodies were used to detect cross-reacting material in locust embryos during early embryogenesis (see Fig. S1E), and indicated that RXR transcripts seen early in embryonic development (20) were translated and therefore of likely functional significance. In vertebrates, activated RXR (as opposed to heterodimerized RXR), is important for neurological promotion in embryos (25, 26). Of the various forms of vertebrate RXRs, RXRα is thought to be of the greatest functional significance during mouse embryogenesis (27).

Ligand Binding and Retinoids.

To evaluate whether Locusta RXR has a role similar to that of the vertebrate receptor during development, it was crucial to determine whether it could bind ligand. Purified, recombinantly expressed locust RXRs competitively bound 9-cis-RA (IC50 = 61.2–107.7 nM) similar to the high affinity shown by hRXRα(DE) in our assays (Fig. 1A and data not shown) and as previously published (22). It is noteworthy that although RXRs from vertebrates have been reported to bind 9-cis-RA (ref. 28 but see also refs. 29 and 30), USP from Dm does not (6). USP may function solely as a dimerization partner for EcR (6, 16, 31). Unlike LmRXR-L, a potential capping loop between helices 1 and 3 of LBD appears to be missing in LmRXR-S, which initially suggested that the small locust isoform might not bind ligand. However, both isoforms showed high affinity for 9-cis-RA. Among invertebrates, only the RXRs from the jellyfish, Tripedalia cystophora, and the sea snail, Biomphalaria glabrata, have been previously shown to bind this ligand (32, 33). We have shown the binding of LmRXR to a ligand, 9-cis-RA, in terrestrial invertebrates.

In chordates, the active vitamin A metabolite, all-trans-RA binds the chordate-exclusive RARs. The Dm genome does not contain an RAR gene (34), and no RAR has been identified in any invertebrate. Indeed, phylogenetic analysis compellingly argues that RARs evolved from duplication events that took place at the origins of vertebrates (35). Nevertheless, both LmRXR-L(DE) and LmRXR–S bind all-trans-RA with high affinity, indicating a role for the receptors in transducing the signal of both retinoid isomers and suggesting that all-trans-RA and 9-cis-RA could act as morphogens in these primitive insects.

Another potential ligand, the polyunsaturated fatty acid, DHA, has been detected in the mouse brain and activates mammalian RXRα with micromolar affinity (36, 37). Similarly, DHA bound competitively to both LmRXR-L(DE) and LmRXR–S isoforms but at a lower affinity compared with the retinoids. As well, the JH analogue, methoprene acid, binds to the locust RXRs with approximately the same low affinity as it does to hRXRα(DE) (Fig. 1) (38). Thus, it appears that locust RXRs are retinoid-specific receptors and unliganded partners for EcR.

To determine whether RAs were present in locust embryos at a time when RXR transcripts were detected, retinoid-sensitive reporter cells (24) were used. When whole locust embryo homogenates were incubated with these cells no significant reporter activity was detected. As well, the addition of homogenates to known concentrations of retinoids lowered the apparent reporter activity (results not shown), suggesting that locust homogenates interfered with the cell line assay. Therefore, efforts were made to extract retinoids from the early embryos.

Although procedures for the extraction of retinoid isomers have been previously described for many vertebrate embryo types and tissues including mouse (39), Xenopus (40), and chicken (41), Locusta embryo extracts presented additional challenges. The main difficulty was the presence of a viscous brown oil, presumably derived from the yolk, which readily precipitated as reported previously for eggs of another locust, Schistocerca gregaria (42). Notably, those authors reported that the presence of the thick oil prevented analysis by HPLC. We were able to circumvent this problem by performing a second, solid-phase extraction followed by tandem MS. Even with a limited amount of insect tissue for extraction, the addition of the preparation to the mouse cells bearing two synergistic endogenous RA response elements (RAREs) (24) was positive; the Cyp26A1 promoter-induced luciferase activity indicated the presence of retinoids. Because this particular cell line is responsive to both 9-cis-RA and all-trans-RA (Fig. 2), these experiments indicated that locust embryos contain measurable levels of retinoids (1.6 ng/g) similar to those reported in mouse serum (0.7–1.1 ng/ml) (43). The subsequent assignment of 9-cis-RA and all-trans-RA as the embryonic retinoids was achieved by their retention time on RP- HPLC and by the specific 299–255 fragment because of a loss of the carboxyl group as determined by tandem MS, derived from RA isomers. Thus, this unambiguous determination of 9-cis-RA in any embryo could only be accomplished by using highly sensitive HPLC-MS/MS in the MRM mode.

Previously, all-trans-RA, and its precursor, all-trans-retinol, has been identified in several vertebrate tissues and embryos (e.g., refs. 39 and 4447). There have also been several reports that there are insignificant levels of 9-cis-RA in these same species (48, 49). Indeed, some have suggested that 9-cis-RA is not the endogenous ligand for RXR (28, 29). However, Luria and Furlow (26) argued that 9-cis-RA was a “logical candidate” for the RXR ligand but that DHA could also be the important morphogen in Xenopus. Here, we show that DHA does not bind to locust RXR with such high affinity compared to 9-cis-RA and that 9-cis-RA is present, representing ≈55% of the retinoids in the locust embryo. As in mammals where compartmentalization of RAs can occur (50), we expect that 9-cis-RA and all-trans-RA would not be evenly distributed throughout the egg. Certainly by day 5 approximately half the egg is yolk, with the embryonic insect occupying the posterior portion. Nevertheless, the presence of RA (≈3 nM) coinciding with the presence of RXR (Kd = 3.0 nM) at a period of rapid embryonic growth just before dorsal closure and the production of 20-OH-Ec and JH by developing prothoracic glands and corpora allata, respectively (51, 52), argues for its importance during development.

Although the role of 9-cis-RA in vertebrates remains unclear, the significance of this isomer as an important signaling molecule in invertebrates is better established. 9-Cis-RA has been detected in adults of the cephalochordate, amphioxus, Branchiostoma floridae (53), and the limb blastemas of an arthropod, the crab, Uca pugilator (54). Furthermore, 9-cis-RA acts as a potent morphogen in the gastropod, Thais clavigera, where exogenous 9-cis-RA induced male genital tracts in females (55). In insects, exogenous all-trans-RA inhibits metamorphosis and arrests embryogenesis (56). Therefore, our identification of 9-cis-RA in Locusta embryos argues strongly that RXR and this natural ligand are also active morphogens in primitive insect embryogenesis. Thus locusts may be a good system to investigate the mode of synthesis of this retinoid, which is still not clear (Table 2) (57). We further speculate that similar to the function of all-trans-RA in vertebrate anterior-posterior patterning mechanisms, 9-cis-RA may also have a role in early mesoderm patterning in invertebrates along with a primitive signaling pathway. Therefore, locust RXR may not merely serve as a silent partner with EcR as does the USP of flies and moths, but it reasonably has a major role in early embryonic development. Indeed, this role is likely ancestral and conserved (57, 58) in invertebrates, but may have been partially usurped by the loss of retinoid binding by the USPs of the more advanced insects and the evolution of RARs in vertebrates (Table 2).

Table 2.
A summary of retinoid synthesis and signaling pathways in mammals and insects

Experimental Procedures

Animals.

African migratory locusts, L. migratoria migratorioides, were reared under gregarious colony conditions, which are standard for our experiments (59). Reproductively mature female locusts oviposited into plastic cups lightly packed with wet sand. Cups were replaced daily and embryos were kept at 31°C where first-instar larvae emerged on day 14. Embryos were recovered from egg pods in the sand cups at day 5 and stored at −80°C.

Plasmids, Protein Purification, and Western Blotting.

The full-length isoform LmRXR-L (GenBank accession no. AY348873) was transformed into M15[pREP4] cells (Qiagen), but also subcloned to generate the LBD (domain DE) for recombinant expression and functional studies. LmRXR-L (corresponding to Thr-176 to Ser-411) was PCR-amplified, subcloned, and expressed (see SI Text). The sequence corresponding to the DE domain was then obtained by restriction enzyme digestion and used to transform BL21(DE3) cells (Novagen) by using pET-15b as a vehicle. The resulting LmRXR-L(DE) construct was sequenced twice in both directions to ensure veracity.

LmRXR-S (GenBank accession no. AF136372) in M15[pREP4] has been described (19). Both LmRXR-S and LmRXR–L constructs were designed to produce recombinant locust proteins bearing six His residues on the amino-terminal end. The hRXRα(DE) (GenBank accession no. NM002957) also with His residues has been previously used for crystallographic studies (60). E. coli cells [LmRXR-L(DE) and hRXRα(DE)] and M15[pREP4] (LmRXR-L and LmRXR-S) were used for the production of recombinant RXRs by using standard procedures (see SI Text). The purified proteins were used to generate antibodies for Western blots (see SI Text) and ligand binding assays.

Ligand Binding Assay.

Displacement binding experiments were performed according to Allegretto (61). Briefly, LmRXR-L(DE) and LmRXR-S purified as described (see SI Text) were incubated in borosilicate glass tubes containing binding buffer [0.15 M KCl, 10 mM Tris·HCl (pH 7.4), 8% (vol/vol) glycerol, and 0.5% (wt/vol) CHAPS detergent] with ≈5 nM [3H]-9-cis-RA (1.44 TBq/mmol or 39.0 Ci/mmol; Amersham) to a final volume of 300 μl. Purified, recombinant Dm dihydrofolate reductase was used as a control protein. Competitor ligands were dissolved in 100% (vol/vol) ethanol and added in increasing concentrations and incubated for 4 h at 4°C. Competitors included 9-cis-RA (0.046–50,000 nM), all-trans-RA (0.042–42,000 nM), methoprene acid (3.1–310,000 nM), and cis-4,7,10,13,16,19-DHA (1.3–1,300,000 nM). Hydroxylapatite (HAP; Bio-Rad) was used to separate bound from free ligand (62). Three-hundred microliters of HAP slurry (0.1 mg/ml) was added to the binding reactions and incubated for 45 min on ice (with mixing every 5 min). The samples were then diluted with 0.9 ml of binding buffer and centrifuged at 857 × g for 5 min at 4°C. The HAP pellet was washed three more times and transferred to a scintillation vial with 0.5 ml of binding buffer and 4.5 ml of liquid scintillant (Amersham Canada). Saturation binding experiments using LmRXR-L(DE) were performed with increasing concentrations of [3H]-9-cis-RA in the presence and absence of 300-fold unlabeled 9-cis-RA. Specific binding, determined by subtracting nonspecific binding from total binding, was subjected to Scatchard analysis. Each sample was counted (Beckman LS6500 liquid scintillation counter), and the binding constant and IC50s were calculated by using GraphPad Prism 3.0 software.

Luciferase Assays.

A P19 mouse embryomal carcinoma cell line was transfected with pGL3-Basic (Promega) containing a 2.6-Kb Cyp26A1 promoter sequence (Cyp26A1 is a member of the cytochrome P450 gene family) fused to a firefly luciferase reporter gene (24). The cells were seeded in 24-well plates at a density of 30,000 cells per well and incubated for 24 h at 37°C in 5% CO2. The cells were then washed with 1 ml of 1× PBS and treated with 0.5 ml MEM (pH 7.3, supplemented with 0.37% NaHCO3, 10% FCS, 0.5% penicillin-streptomycin, 0.1% gentamicin, and 0.1% fungizone; Invitrogen Life Technologies). The MEM was supplemented with DMSO, all-trans-RA, a mixture of all-trans-RA and 9-cis-RA, day-5 Locusta embryo homogenates, or extracts in the amounts listed in Results. The cells were incubated for an additional 24 h, washed as before, and lysed with 200 μl of passive 1× lysis buffer (Promega) for 20 min. Lysates were scraped from the bottom of the wells, 20-μl samples were treated with luciferase reagents (Promega), and enzyme levels were determined immediately in a Berthold luminometer. Data were analyzed by using Student's t test.

Embryo Retinoid Extraction.

All dissections, extractions, and downstream analyses were performed under dim amber lighting, and glassware was used in sample preparation and handling. Day-5 locust embryos were ground in homogenization buffer (PBS containing 100 μM 1,2-dianilinoethane) for 2 min by using a Rotor Stator homogenizer (KikaWerk). Embryo samples included 3 g of embryos, 1.5 g of embryos with 1.5 g of BSA, and 3 g of BSA, all in triplicate; all samples were extracted according to a modified Bligh and Dyer (23) protocol. Homogenates were adjusted to pH 9.5 to partition the deprotonated RA to the aqueous phase and nonpolar lipids to the organic phase. The homogenates were extracted with 2 × 5 ml 100% (vol/vol) methanol, 2 × 5 ml dichloromethane, and 5 ml of saturated KCl and centrifuged at 1,000 × g at 4°C for 15 min. The aqueous phase was removed with a Pasteur pipette and subsequently dried in a sample evaporator (37°C) under a gentle stream of N2 to reduce the content of organic solvent. Samples were resuspended in 1 ml of 5% (vol/vol) methanol, vortexed, and subjected to a second solid-phase extraction. Waters Oasis hydrophilic-lipophilic balanced cartridges (6 ml) were washed with 6 ml of 100% methanol and then 6 ml of ddH2O. Fractions were collected by vacuum filtration, washed once with 6 ml of 5% (vol/vol) methanol, and eluted with 6 ml of 100% (vol/vol) methanol. Eluants were dried in a sample evaporator (37°C) under a gentle stream of N2, resuspended in 1 ml of 100% (vol/vol) ethanol, dried again, and resuspended in 30 μl of 60% (vol/vol) acetonitrile. Sample standards (500 ng of all-trans-RA and 500 ng of 9-cis-RA) were dried under N2 and resuspended in 30 μl of 60% (vol/vol) acetonitrile. Standards and samples were centrifuged for 15 min at 1,500 × g at 4°C and transferred to glass vials with a polyspring insert for HPLC analysis. Concentrations were calculated by using the formula weight of all-trans-RA and 9-cis-RA (300.44 g/mol) and the embryonic volume of Locusta (0.01 ml).

HPLC and MS.

HPLC/MS analysis of extracted locust embryos was performed as described by Chithalen et al. (63). Briefly, RP-HPLC separation of extracted samples was performed on a Waters 2695 separation module with a 996-photodiode array (monitoring at 350 nm) and a Zorbax-SB C18 column (Agilent; 3.5 μm, 150 × 2.1 mm) by using a gradient solvent system of ddH2O-acetonitrile-glacial acetic acid [74.99:25:0.01 (vol/vol/vol) to 0.99:99:0.1 (vol/vol/vol) at a flow rate of 200 μl/min for 35 min with final conditions held for 5 min]. Samples were further analyzed by the most sensitive LC-MS/MS MRM system available to us. A Micromass Quattro Ultima (Waters) mass spectrometer (equipped with a Z-Spray electrospray interface in the negative mode set with a capillary voltage of −3.70 kV and a cone voltage of −55 V) was used. The source and desolvation temperatures were maintained at 80°C and 350°C, respectively, and the cone and desolvation gases were kept at 50 and 562 liters/h, respectively. Argon, used as the collision gas, was maintained in the chamber at 1.6 × 10−3 mBar with a collision energy of 14 V. MRM was used to observe the transition of m/z 299–255. The area under the curve was calculated for each chromatogram and averaged. Isomer standards (including radiolabeled 9-cis-RA) were used, and all-trans-RA, 9-cis-RA, and 13-cis-RA were run both before and after each of the samples to ensure there was no drift in retention times and no isomerization of the retinoids. The LC/MS was run by using single reaction monitoring so that the transition from 299 to 255 (loss of a carboxyl group) was detected, ensuring a specificity for RA.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank Drs. S. Zhou, D. Hayward and E. Ball for their encouragement, Dr. H. Gronemeyer (Institut de Génétique et de Biologie Moléculaire et Cellulaire/CNRS/INSERM/Université Louis Pasteur, Strasbourg, France) for the generous gift of the hRXRα(DE), the reviewers for their suggestions. The majority of this work was supported by a Natural Sciences and Engineering Research Council (Canada) grant (to V.K.W.), and Canadian Institutes of Health Research Operating and Equipment Maintenance Grants MT-9475 and SMFSA-260938 (to G.J.) are also acknowledged.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. W.S.L. is a guest editor invited by the Editorial Board.

This article contains supporting information online at www.pnas.org/cgi/content/full/0712132105/DCSupplemental.

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