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Intracellular localization and tissue specificity of the
Methoprene-tolerant
(
Met
) gene product in
Drosophila melanogaster
Stephen Pursley, Mudgapalli Ashok, Thomas G. Wilson
*Department of Biology, Colorado State University, Fort Collins, CO 80523, USA
Received 31 October 1999; received in revised form 31 December 1999; accepted 25 January 2000
Abstract
TheMethoprene-tolerant(Met) gene product inDrosophila melanogasterfacilitates the action of juvenile hormone (JH) and JH analog insecticides. Previous work resulted in the cloning and identification of the gene as a member of the bHLH-PAS family of transcriptional regulators. A Met+ cDNA was expressed in Escherichia coli, and polyclonal antibody was prepared against the purified protein. A single band on a Western blot at the expected size of 79 kD was detected in extracts from Met+larvae but not from Met27null mutant larvae, demonstrating the antibody specificity. Antibody detected MET in all stages of D. melanogaster
development and showed tissue specificity of its expression. MET is present in all cells of early embryos but dissipates during gastrulation. In larvae it is present in larval fat body, certain imaginal cells, and immature salivary glands. In pupae it persists in fat body cells and imaginal cells, including abdominal histoblast cells. In adult females MET is present in ovarian follicle cells and spermathecae; in adult males it is present in male accessory gland and ejaculatory duct cells. In all of these tissues MET is found exclusively in the nucleus. Some of these tissues are known JH target tissues but others are not, suggesting either the presence of novel JH target tissues or another function for MET. 2000 Published by Elsevier Science Ltd. All rights reserved.
Keywords: Metgene; Juvenile hormone; Methoprene; PAS protein; Insecticide resistance
1. Introduction
Identifying the various roles for juvenile hormone (JH) in dipteran insects and elucidating the mechanism of action continue to challenge insect physiologists. In
Drosophila melanogaster, JH has been shown to func-tion in adults during oogenesis (Postlethwait and Weiser, 1973; Soller et al., 1999), larval fat body histolysis (Postlethwait and Jones, 1978), and male accessory gland protein synthesis (Yamamoto et al., 1988). How-ever, progress toward elucidating JH function in preadult dipteran insects has been frustratingly slow. The classi-cal endocrinologiclassi-cal methodology of allatectomy and hormone replacement, which revealed the involvement of JH during preadult development in insects from other orders, has been unsuccessful in Diptera because of the close association of the corpus allatal cells with unre-lated cells in the ring gland (King et al., 1966).
* Corresponding author. Fax:+1-970-491-0649.
E-mail address:[email protected] (T.G. Wilson).
0965-1748/00/$ - see front matter2000 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 0 ) 0 0 0 5 6 - 4
Likewise, elucidating the mechanism of action of juv-enile hormone also has been difficult. A major impedi-ment to understanding how this hormone works is the elusive nature of the JH receptor (Riddiford, 1994; Jones, 1995). Although biochemical approaches have identified intracellular JH binding proteins, some with characteristics expected of a receptor, it has been diffi-cult to relate these proteins to a biological role for the hormone. In our laboratory we have focused on ident-ifying the JH receptor machinery by employing a genetic approach with D. melanogaster. In this approach, we have made use of the high toxicity of methoprene, a JH agonist insecticide (Staal, 1975), to D. melanogaster
demon-strated for several insecticide-resistant insects that have a “target-insensitivity” mechanism of action resulting from a genetically altered target molecule for the insecti-cide (Taylor and Feyereisen, 1996).
To identify methoprene-resistant mutants, progeny from mutagenized methoprene-susceptible D. mel-anogaster were raised on a dose of methoprene that is toxic to susceptible flies (Wilson and Fabian, 1986). Sev-eral resistant mutants were isolated, all of which proved to be alleles at a locus we termed Methoprene-tolerant
(Met). Biochemical studies ruled out resistance mech-anisms of either reduced cuticular penetration or enhanced metabolism, and demonstrated Met flies to possess a high-affinity JH intracellular binding protein that has a 10 times lower binding affinity for JH III than that from Met+ flies (Shemshedini and Wilson, 1990). We have presented indirect evidence that this binding protein is a JH receptor (Shemshedini et al., 1990), but direct evidence is lacking. We believe that theMet+gene may encode either the receptor or a protein that is inti-mately and stoichiometrically involved in JH reception. In previous work we clonedMet+by transposon tag-ging and found the gene to show homology to the bHLH-PAS family of transcriptional regulators (Ashok et al., 1998). This gene structure is consistent with the findings of JH regulation of gene expression in a variety of insects (Jones, 1995). This family includes the aryl hydrocarbon (dioxin) receptor (AHR) and its partner AH nuclear translocator (ARNT), the clock genes and the hypoxia-inducible factor gene (Crews, 1998; Ponting and Aravind, 1998), all of which respond to external sig-nals to the cell.
The intracellular location and tissue specificity of MET could shed light on the action of JH as well as suggest novel roles for the hormone, depending on the tissue specificity. For these studies, antibody was gener-ated to MET that was isolgener-ated following expression of
Met+in a prokaryotic expression system. Here we show that MET is found exclusively in the nucleus of the cell. It is found in all stages of development and localizes to specific tissues, some of which are known JH target tissues.
2. Materials and methods
2.1. Fly stocks and culture
The vermilion (v) strain (obtained from the Mid-American Drosophila Stock Center, Bowling Green, OH) was used as theMet+strain in these as in previous studies (Wilson, 1996; Wilson and Ashok, 1998). The
Met27 allele utilized was recovered from a gamma-irradiation mutagenesis screen of a v parental stock (Wilson and Ashok, 1998). All flies were raised at 25°C
on a standard agar–yeast–cornmeal–molasses diet with propionic acid added to retard fungal growth.
2.2. Microscopy
Stained samples were observed using the Provis AX70 Research System Microscope from Olympus with auto-matic photomicrograph system. DAPI staining was observed utilizing epifluorescent objectives at 515 nm.
2.3. Antibody production
A Met+ cDNA was isolated from a D. melanogaster
ovary cDNA library as previously described (Ashok et al., 1998) and subcloned into the pET-23A expression vector. Maintenance of the reading frame of this cDNA fragment at the vector–insert junction was verified by DNA sequencing. The Met+ cDNA was expressed, and the MET fusion protein purified by SDS–polyacrylamide gel electrophoresis (PAGE). Following verification of molecular weight of the expected fusion protein, it was then extracted from the SDS–PAGE gel, injected into New Zealand white rabbits, and followed up with a 30 day booster injection. Antibody was allowed to develop for 96 days. The antiserum obtained was further purified using an Affigel-10 protein A column (Biorad).
2.4. Western analysis
2.5. Tissue fixation and immunostaining
Both larvae and adults were dissected in phosphate buffered saline (PBS) and fixed in PEM-FA as described (Patel, 1994) with the following modifications. Each step in the protocol was performed at 22°C in 1.5 ml microcentrifuge tubes. Tissues were fixed for 1 h, washed with PBT [500 mM 1X PBS, 8% Triton X-100, 5% bovine serum albumin (BSA)] and blocked overnight with PBT+NGS (10%). Tissues were then incubated with primary antibody for 1 h (1:200 dilution) followed by three 1 h washes with PBT. Tissues were then stained with a peroxidase-conjugated goat anti-rabbit IgG (Vector Laboratories) using a 1:1000 dilution for 1 h. Samples were developed using a peroxidase substrate kit (Vector Laboratories). After staining, the tissues were rinsed with PBS and mounted for viewing. Egg cham-bers and embryos were stained as described (Patel, 1994). Several preparations were counterstained with the DNA-specific dye DAPI (4µg/ml) to specifically detect DNA within the nuclei. Control experiments with pre-immune serum were conducted using the same staining procedures as above but using a 1:25 dilution.
3. Results
3.1. Production and specificity of antibody
MET antigen for antibody production was produced by expressing aMet+cDNA molecule. When introduced into Met flies by germline transformation, this cDNA was shown to rescue the resistance phenotype (Ashok et al., 1998), thus demonstrating that it encodes a func-tional MET protein.
Following expression ofMet+inEscherichia coli, the bacterial cells were homogenized, and the proteins were separated by SDS–PAGE. An intense protein band cor-responding to the expected molecular weight was excised and used as antigen source for antibody prep-aration. This antibody was used in a Western blot analy-sis ofMet+larval extract. As shown in Fig. 1(A), follow-ing SDS–PAGE separation of proteins from whole larvae, a single band was detected on a Western blot. The molecular weight of this band agreed with the pre-dicted size of MET of 78,683 calculated from the deduced amino acid sequence (Ashok et al., 1998). Importantly, this band was absent in flies homozygous for Met27, which is a null allele of Met+ (Wilson and Ashok, 1998). Therefore, the antibody is specific for MET.
3.2. Intracellular location of MET
To identify the intracellular location of MET, we first examined larval fat body, a tissue previously shown to
be a target tissue for JH (Postlethwait and Jones, 1978) and to reflect the altered JH binding seen in Met flies (Shemshedini and Wilson, 1990). Late third-instar larvae were used as a source for this tissue. Incubation with antibody followed by horseradish peroxidase detection revealed intense staining in the nucleus [Fig. 1(C)]. No other portion of the cell showed staining. Met27 larval fat body failed to show MET staining, confirming the specificity of the antibody [Fig. 1(C)]. To confirm that we were detecting MET in the nucleus, several tissue preparations were stained with DAPI, a DNA-specific stain. Dual viewing of these preparations for MET and for DNA showed the same location in the cells [shown for follicle cells in Fig. 3(C) and (D)]. Therefore, MET is located exclusively in the nucleus of this tissue and in all other tissues to be described that showed staining.
3.3. Tissue specificity of MET
A developmental Northern blot ofMet+flies showed
Met+transcript to be present during all stages of devel-opment (Ashok et al., 1998). Therefore, we hypothesized that MET is present throughout development. To test this hypothesis and to identify the tissues containing MET, permeabilized embryos and dissected postembryonic stages were fixed and subjected to immunostaining with
Met+antibody.
3.3.1. Embryo
MET stained intensely in all cells from about the 256-cell stage until early gastrulation [Fig. 1(B)], when the staining gradually became less intense with the pro-gression of embryonic development. Late-stage embryos showed regions of diffuse light staining, but it could not be readily localized to specific embryonic tissues (not shown). The results show that MET is present during early embryogenesis but suggest that its production may not continue as gastrulation proceeds, thus resulting in a dilution of staining intensity.Met+embryos stained with preimmune serum served as controls and showed no staining [shown in Fig. 1(B) for the 256-cell stage], as expected.
3.3.2. Larvae
Fig. 1. Expression of MET and localization in embryonic and larval tissues. (A) Western blot of third-instar whole larval extracts ofMet+and
Met27separated by SDS–PAGE and blotted with MET antibody. The arrow shows the location of the band found inMet+. On this particular blot a modicum of staining is evident in theMet27lane, which we interpret as non-specific activity because it was not evident either on other Western blots or from theMet27tissue immunostaining results. (B) Immunostaining ofMet+early embryos with either preimmune serum or MET-derived
antibody. The localization of MET is strictly nuclear. (C) MET localization inMet+andMet27larval fat body from late third-instar larvae. Note the nuclear localization. (D) Localization of MET in larval salivary glands of aMet+third-instar larva exhibiting non-synchronous gland develop-ment. The less-developed gland (small arrow) shows MET expression, while the transparent well-developed gland (large arrow) shows the loss of MET typical of this stage in larval development.
imaginal disks (Fig. 2). In imaginal disks the staining was uniformly distributed.
3.3.3. Pupae
Because of tissue histolysis during pupal develop-ment, detection of MET was more difficult. During the first 24 h of pupal development, it immunolocalized in the imaginal tissues described above and in the larval fat body, but the staining intensity diminished with time, similar to its appearance in embryos. It was also found in the abdominal histoblast cells [Fig. 2(F)], which are undergoing mitosis and differentiation to form the adult tergite and sclerite structures (Roseland and Schneider-man, 1979). These cells are sensitive to topically applied
JH and JH analog insecticides (Ashburner, 1970; Mad-havan, 1973; Wilson and Fabian, 1986).
3.3.4. Adults
Fig. 2. Expression of MET in late third-instar larval and early pupal tissues. (A) Imaginal cells of the midgut. (B) cells of the hindgut imaginal ring. Note also staining in several of the larval midgut cells. Such midgut larval cell detection of MET was occasionally seen in nuclei in close proximity to the imaginal cells. (C) Cells of the adult salivary gland imaginal ring (arrow). The larval salivary gland cell (larger cells) nuclei are staining intensely. (D) Imaginal cells of the adult tracheae. (E) Wing imaginal disk. (F) Abdominal histoblast cells in a 1 day pupa. Note the absence of MET staining in the large larval epidermal cells, whose nuclei are DAPI-stained in this preparation.
In males the male accessory gland and ejaculatory duct [Fig. 3(A)] cells stained intensely.
4. Discussion
It is clear from previous work that the Met gene is involved in facilitating the response of D. melanogaster
to JH and JH analog insecticides (Wilson and Fabian, 1986; Riddiford and Ashburner, 1991; Restifo and Wil-son, 1998). The immunolocalization of MET has helped to better understand the role of MET in this process in two ways: (1) it has demonstrated that MET is found only in the nucleus of cells expressingMet+, presumably where it functions; and (2) it has shown Met+ to be expressed both in tissues shown in other work to be JH target tissue (e.g., male accessory glands) as well as in tissues not known to be JH target tissue (e.g., embryo). First, MET is found in the nucleus. This result rules out a possible interpretation of MET function as a cyto-solic JH binding protein “sink”, similar to the cytocyto-solic retinoic acid binding protein in vertebrate cytosol (Li, 1999). In our previous work we detected JH III binding in the cytoplasm of larval fat body (Shemshedini et al.,
1990; Shemshedini and Wilson, 1990) and photoaffinity analog labeling in both cytosolic (Shemshedini et al., 1990) and nuclear (Shemshedini and Wilson, 1993) frac-tions of D. melanogaster cells, but in the present study MET was not detectable in the cytoplasm. We have two possible explanations for this discrepancy: (1) the cyto-plasmic preparations in the previous biochemical studies were contaminated with nuclear material; or (2) the MET protein is not the JH-binding component of the JH tran-scriptional regulatory machinery. Since bHLH-PAS pro-teins usually function as heterodimers (Ponting and Ara-vind, 1998), it is possible that the JH binding difference seen between Met and Met+ preparations (Shemshedini and Wilson, 1990) is due to the partner of MET.
Fig. 3. Expression of MET in adult reproductive tissues ofMet+flies. (A) Immunolocalization in the male accessory glands (small arrow) and ejaculatory duct (large arrow). Staining is largely absent in the testes (dotted arrow). (B) Immunolocalization in the spermathecae. (C) Immunolocal-ization in the follicle cells surrounding a stage-10 egg chamber. Those follicle cells surrounding the nurse cells are considerably spread compared with those surrounding the oocyte. (D) DAPI staining in a similar-stage egg chamber demonstrating the nuclear localization of MET. The large nurse cell nuclei, which do not stain with MET antibody, are DAPI-stained.
casts doubt on a mechanism of action that precisely mir-rors that of AhR–ARNT.
Second, MET is found in a variety of tissues. Some of these are known JH target tissues. These include the male accessory gland and ejaculatory duct (Yamamoto et al., 1988; Wyatt and Davey, 1996), the larval fat body (Postlethwait and Jones, 1978), and the follicle cells of vitellogenic oocytes (Soller et al., 1999). Other tissues are not suspected JH targets, however, including the developing larval salivary glands, early embyonic cells, and the imaginal cells that showed MET during preadult development. The latter finding means either that MET has an additional function(s) in flies other than facilitat-ing JH action or that these tissues should be regarded as possible JH targets. However, the present work cannot distinguish between these latter two possibilities. It was hoped that the phenotype of theMet27null mutant would help illuminate additional roles for JH in flies, but it has not. The phenotype is milder than one might expect from loss of a JH receptor (or associated) protein: although the flies are generally less vigorous than wild-type, they are viable, demonstrating thatMetis not a gene required for viability. Clouding the interpretation of the Met27 phenotype is “genetic redundancy” seen for many genes in both vertebrates and invertebrates (Brookfield, 1997):
important genes have a backup gene or mechanism that rescues a phenotype perilous to the organism when the gene is mutated. The most dramatic phenotypic character in Met27was a reduction in oogenesis to about 20% of that of wild-type, possibly by an interruption in the JH control of this process (Wilson and Ashok, 1998). Cer-tainly, this phenotype is consistent with the finding of MET in the follicle cells of vitellogenic oocytes.
The detection of MET in imaginal cells is a surprise. A variety of imaginal cell types were seen to show MET, including adult salivary gland cells, foregut, midgut, and hindgut imaginal cells that are slated to replace the pre-adult gut cells in the pre-adult, tracheal imaginal cells which will form adult tracheal tubes, and finally imaginal disk cells which form the cuticular structures in the adult fly. Although no role for JH has been shown in these imaginal cell types, it is tempting to hypothesize that JH may maintain the “status quo” (Riddiford, 1996) of these cells until ecdysone-induced differentiation occurs.
possibilities: (1) MET has an additional function(s) in flies other than facilitating JH action; (2) unidentified additional roles for JH exist in all stages of development, necessitating a more widespread expression of MET than predicted from extant roles; or (3) MET functions as a partner for the “real” JH receptor, similar to ultraspiracle (usp) as the ecdysone non-binding partner of EcR that is expressed throughout development (Andres et al., 1993). The present work cannot distinguish among these possibilities.
PAS proteins commonly function as heterodimers with other PAS proteins (Ponting and Aravind, 1998). When the putative partner protein of MET is identified, perhaps some of these questions can be answered.
Acknowledgements
This study was supported by NSF grant IBN-9419774 to TGW.
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