Tissue & Cell, 1998 30 (2) 187-194 © 1998 Harcourt Brace & Co. Ltd Immunohistochemical localization of ecdysteroid receptor and ultraspiracle in the epithelial cell line from Chironomus tentans (Insecta, Diptera) M. Lammerding-K6ppel 1, M. Spindler-Barth 2, E. Steiner 1, M. Lezzi 3, U. Drews 1, K.-D. Spindler' Abstract. Ecdysteroid receptor (EcR) and its heterodimerization partner, ultraspiracle (USP), were demonstrated in the epithelial cell line from Chironomus tentans by immunohistochemistry. In untreated cells both proteins are present in nuclei as well as in granular compartments of the cytosol. At 1 day after addition of 1-pM 20-OH-ecdysone (20E) total immunofluorescence had increased in the nuclei, whereas the cytoplasmic staining had disappeared. At the 2nd and 3rd days all cells within a vesicle appear identical according to morphological criteria, but the EcR and USP immmunoreactivity becomes restricted into patches of neighbouring cells. The hormonally induced changes in the pattern of localization of functional ecdysteroid receptor, the heterodimer of EcR and USP, are discussed in relation to similar effects of 20E on acetylcholinesterase and muscarinic acetylcholine receptor distribution in this cell line. Keywords: Heterodimerization,ecdysteroidreceptor,ultraspiracle, insect, cell line Introduction The permanent epithelial cell line from the dipteran Chironomus tentans responds to moulting hormone with a broad variety of physiological and morphological effects. The affinity of ecdysteroids to ecdysteroid receptors (EcRs) correlates with their biological effects, as demonstrated by the induction of acetylcholinesterase (Spindler-Barth, 1991; Spindler-Barth et al., 1997), and chitinase (Quack et al., ~Anatomisches Institut der Universit&t TiJbingen, Osterbergstr. 3, D-72074 T~3bingen, Germany. 2Lehrstuhl ft~r Hormon- und Entwicklungsphysiologie, Heinrich-HeineUniversitY.t, Dfisseldorf, Universitb.tsstr. 1, D-40225 DOsseldorf, Germany. 31nstitut ffir Zellbiologie, ETH Z0rich, H6nggerberg, CH-8093 ZiJrich, Switzerland 4Abteilung AIIgemeine Zoologie, Universit&t UIm, AIbert-Einstein-Allee 11-13, D-89069 UIm, Germany. Received 2 May 1997 Accepted 4 December 1997 Correspondence to: Klaus-Dieter Spindler, Abteilung AIIgemeine Zoologie, Universit&t UIm, A[bert-Einstein-Allee 11-13, D-89069 UIm, Germany. Te1:49731 5022583; Fax: 49731 5022581. 1995). As shown previously (Lammerding-K6ppel et al., 1994), changes in cell shape and cell arrangement do not occur in all cells simultaneously. The hormonal response is visible first in cells which possess an enhanced muscarinic receptor concentration and increased acetylcholinesterase activity. Both phenomena are considered to be part of a muscarinic system involved in tissue differentiation in a wide variety of animals (Drews, 1975; Buznikov et al., 1996). Since muscarinic receptor concentration (Wegener et al., 1996) and acetylcholinesterase activity are regulated by 20OH-ecdysone (20E) (Spindler-Barth, 1991; Spindler-B arth et al., 1988) in Chironomus cells, the question arose whether those cells which are engaged in morphogenetic reactions also show changes in ecdysteroid receptor (EcR) concentration and distribution. Since the heterodimer of EcR and ultraspiracle (USP) is considered as a functional ecdysteroid receptor complex (Yao et al., 1993; Segraves, 1994), we wanted to know whether USP co-localizes with EcR and responds to 20E in the same way. On giant chromosomes from the salivary gland of Chironomus tentans USP and EcR co-localize at ecdysteroid-dependent puff sites (Wegmann et al., 1995). 187 18 8 LAMMERDING-KOPPEL ET AL. LOCALIZATION OF EcR AND USP IN THE CELL LINE FROM CH1RONOMUS TENTANS 189 Fig. 1 Immunofluorescent detection of ultraspiracle (USP) with the monoclonal antibody mAB I 1 in multicellular vesicles of Chironomus tentans. Ceils of untreated vesicles show faint immunofluorescence in the nuclei and, in addition, stronger granular fluorescence in the cytoplasm. (A) An FITC-labelled second antibody was used. Long exposure epifluorescence micrograph (20 s). Inset: digital magnification of 200%. (B) Digital contrast enhancement to demonstrate immunofluorescence in cytoplasmic vesicles. (C) Phase contrast of the same vesicle, x 360. Fig. 2 USP immunofluorescence after 24 h of treatment with 1 gM 20-OH-ecdysone. All nuclei are strongly stained. The fluorescence intensity was much higher than in untreated vesicles resulting in shorter exposure times during microphotography. Immunofluorescent granules are no longer detectable in the cytoplasm. (A) An FITC-labelled second antibody was used. Epifluorescence micrograph of whole mount vesicle (exposure time 10 s). Inset: digital magnification of 200%. (B) Digital contrast enhancement as in Fig. lB. (C) Phase contrast of the same vesicle. × 360 Materials and methods Cell culture The epithelial cell line from Chironomus tentans established by Wyss (1982) was kept in suspension culture in T flasks at 25°C in a medium containing 2% fetal calf serum. The cells grow as multicellular spherical vesicles of 100 to 300 gm diameter, consisting of one layer of flat cells surrounding a liquid-filled cavity. Propagation and hormonal treatment of the cells were performed as described previously (Lammerding-Ktppel et al., 1994). Immunohistochemistry Vesicles were harvested by centrifugation (5 rain, 1000 r.p.m), washed with phosphate-buffered saline (PBS; 137mM NaC1, 2.7mM KC1, 8raM K2HPO4, 1.5raM KH2PO4, pH 6.7) and fixed in 4% formaldehyde (freshly made from paraformaldehyde) in PBS for 15 rain at 4°C. Fixation was stopped by addition of ice-cold PBS, followed by centrifugation. The pellet was rinsed first for 5 rain in PBS, for 15 min in PBS containing 50 mM NH4C1, for 5 rain in 300 gl of methanol (- 20°C), for 5 rain in acetone (- 20°C) and finally washed in PBS (pH 7.4). Fixed vesicles were preincubated for 20 rain at room temperature in PBS (pH 7.4), containing 2% bovine serum albumin and 0.1% Triton X-100 (both final concentrations), followed by incubation with either a polyclonal antibody against the D-domain of the Chironomus tentans EcR (Wegmann et al., 1995; final dilution 1:4) or with the monoclonal antibody mAB 11 against Drosophila melanogaster USP (final dilution 1:50). Incubations were for 3 h at room temperature for anti-EcR and overnight in the cold for anti-USP. Incubation was stopped by the addition of ice-cold PBS containing 0.1% Triton X-100; the vesicles were centrifuged and the pellet was washed once with the same buffer. Cells were incubated for 1 h with Texas Red-labelled goat anti-rabbit IgG (final dilution 1:350; Dianova, Hamburg, Germany) for detection of EcR, or an FITC-labelled rabbit anti-mouse IgG (final dilution 1:300; Dianova) for USE Incubations were stopped by the addition of ice-cold PBS (with 0.1% Triton X-100, pH 7.4). After centrifugation the pellet was washed three times for 10 rain and eventually suspended in PBS (pH 7.7). All steps were carried out under continuous shaking. Control experiments were performed by: (1) omitting the primary antibody; (2) substituting normal rabbit serum, and (3) incubation with a non-matching secondary antibody. Results Vesicles of the epithelial cell line from Chironomus tentans show distinct morphogenetic changes after addition of moulting hormone 20E, as depicted schematically in Figure 8. EcR and USP can be detected by immunofluorescence in untreated vesicles, but both the intensity and distribution of fluorescence within the vesicles change after hormone treatment (Figs 1-6). Untreated vesicles All nuclei of untreated vesicles are EcR- and USP-positive but the fluorescence is only weak (Fig. 1A, 3). In addition to the nuclear localization of EcR and USP, both are also found in cytoplasm (Fig. 1B, 3). In approximately 30-40% of the vesicles EcR is clearly visible in dis~tinct granulae in the cytoplasm (Fig. 3). Treatment with 20-OH-ecdysone After addition of 1 gM 20E there is an increase in the intensity of fluorescence for both EcR (Fig. 4) and USP (Fig. 2). The first signs are already noticed after an incubation of 12 h, but are clearly visible after 24 h (Figs 2, 4), when all nuclei are intensively stained with both antibodies and EcR and USP are no longer detected in cytoplasmic granulae. At 2 days after hormone application the morphogenetic processes are visible (Fig. 8). The first groups of neighbouring cells elongate in a centripetal direction; later this reaction spreads over the whole vesicle and the originally squamous epithelium is changed to a columnar, stratified tissue. After incubation with hormone for 48 h most of the nuclei are still intensively stained but there are also some groups of cells with only faint or even negative staining by anti-EcR and anti-USP antibodies. After hormone treatment for 3 days, the originally round vesicles become more and more irregular in shape (Fig. 8) due to the asynchronous changes in cell shape and cell arrangement. For both EcR and USP, intense staining of 190 LAMMERDING-KOPPEL ET AL. 4 5 7A LOCALIZATION OF EcR AND USP IN THE CELL LINE FROM CHIRONOMUS TENTANS 191 Fig. 3 Immunofluorescent detection of ecdysteroid receptors (EcR) with a polyclonal antibody in multicellular vesicles of Chironomus tentans. Untreated vesicles show weak immunofluorescence in the nuclei, and in addition strong and distinct granular fluorescence in the cytoplasm. A Texas Redlabelled second antibody was used. Long exposure epifluoreseence micrograph (20 s). × 360. Inset: digital magnification of 200%. Fig, 4 Immunofluorescent detection of EcR in 20-OH-ecdysone-treated vesicles after 24 h of treatment. All nuclei are intensely stained. No anti-EcR immunoreactivity can be detected in cytoplasmic granules any more. A Texas Red-labelled second antibody was used. Exposure time 10 s. x 360. Inset: digital magnification of 150%. Fig, 5 Immunofluorescent detection of EcR in 20-OH-ecdysone-treated vesicles after 72 h of treatment. In many vesicles there are patches of neighbouring cells with highly fluorescent nuclei which in transmitted light were coincident with stratified areas. Some protrusions of the vesicles possess only two or three fluorescent cells. A Texas Red-labelled second antibody was used. Exposure time 10 s. x 360. Fig, 6 USP immunofluorescence after 72 h of treatment with 1 gM 20-OH-ecdysone. In many vesicles there are patches of neighbouring cells with highly fluorescent nuclei. Some buds of vesicles show only single cells with nuclear immunofluorescence. An FITC-labelled second antibody was used. Exposure time 10 s. x 360. Fig, 7 Control of USP immunoreactivity. (A) Incubation of untreated vesicle with normal rabbit serum instead of primary antibody. FITC fluorescence; exposure time 20 s. x 360. (B) Phase contrast of the same vesicle. nuclei occurs especially in single cells of those areas with 'buds' where the morphogenetic reaction is most advanced. After prolonged hormone treatment, immunostaining of EcR and USP decreases (Figs 5, 6). Discussion Although all cells of the epithelial monolayer which form the multicellular vesicle appear the same according to morphological criteria, the first visible responses to moulting hormones are local (Spindler-Barth et al., 1992, 1995; Lammerding-K6ppel et al., 1994). After prolonged treatment, all cells in the vesicle respond to the hormone. The regions which show the first signs of morphogenetic response are characterized by an overexpression of acetylcholinesterase and muscarinic acetylcholine receptor (Lammerding-KOppel et al., 1994) which may indicate a functional role of the muscarinic system in cell differentiation in the Chironomus cell line. Our results demonstrate that in hormonal naive cells EcR and USP distribution are the same in all cells. Nevertheless the hormonal response starts in small groups of adjacent cells. Since no positional information is possible within the vesicles, endogenous differences between the cells must cause this variability of the hormonal response. Since all cells have the intrinsic capacity to respond to ecdysteroids but the time necessary for achieving their full response varies, the most likely explanation for this behaviour is differences in the cell cycle, as proposed by Hattet al. (1994, 1997). For various insect cell lines it was shown that 20E stops cell division (Wyss, 1980, 1982, Dinan et al., 1990) preferentially at G2 (Stevens et al., 1980; Hatt et al., 1994). In general, the cell division cycle is not synchronized between cells within a multicellular vesicle (Nicolaidis and Spindler-Barth, unpublished observation), which does not exclude the possibility that small groups of neighbouring cells are temporarily synchronized as is described during wing morphogenesis (Bryant, 1996; Milan et al., 1996). Modulation of steroid hormone action during the cell cycle is already described for glucocorticoids (Hsu and de Franco, 1995) and is currently being investigated in Chironomus cells. Enhanced ecdysteroid receptor concentration is the first sign that we observe in cells destined to show morphogenetic reactions, and it precedes the increase in muscarinic receptor concentration. The decrease of immunoreactivity as shown here for EcR and USP coincides with the fall in muscarinic receptor concentration (Lammerding-K6ppel et al., 1994; Wegener et al., 1996). Transient changes in the expression of hormone-dependent genes involved in cell differentiation are also observed for actin and tubulin (Fig. 9). Finally acetylcholinesterase activity rises (Fig. 9). The simultaneous increase of USP in the same cells is in accordance with the fact that the functional ecdysteroid receptor is a complex of EcR and USP (Yao et al., 1993; Segraves, 1994). Induction of hormone receptors by their cognate hormones seems to be a widespread phenomenon, common to many steroid hormones (Tata, 1994). ACKNOWLEDGEMENTS We gratefully acknowledge the kind gift of the monoclonal antibody mAB 11 directed against Drosophila melanogaster ultraspiracle from Dr Kafatos (Heidelberg), as well as the financial support of our work by grants from the Deutsche Forschungsgemeinschaft to M.S.B. and K.D.S. (SFB 351, A5) and from the Swiss Science Foundation to M.L. 192 LAMMERDING-KOPPEL ET AL. 20-OH-Ecdysone treatment Untreated vesicle S Cytoplasmic immunoreactivity of EcR and USP Nuclear immunoreactivity: 0 weak @ medium • strong 72 h t 48 h Fig. 8 Changes in the expression of the functional ecdysteroid receptor, the heterodimer of EcR and USP in the Chironomus tentans cell line after 20-OH-ecdysone (20E) treatment. Since the immunoreactivities of anti-EcR and anti-USP show staining patterns similar in intensity and distribution, they are not distinguished here. Morphogenetic changes in the vesicles proceed without cell proliferation by rearrangement of the cells. After the addition of 20E, immunoreactivity clearly shifts from cytoplasm into the nuclei. Nuclear staining is most intense in protruding areas. It decreases in most cells of the final aggregates. LOCALIZATION OF EcR AND USP IN THE CELL LINE FROM CHIRONOMUS TENTANS 193 20E cell proliferation (I) I EcRt I [ USP 9 -V EcR/USP ® ® // ,, [ actin,tubulin (4) ImAchR (3) I AchE (5)i Imorphogeneticresponse(6)1 Fig. 9 The events taking place between formation of the functional ecdysteroid receptor complex and the ecdysteroid-indnced morphogenetic processes in the Chironomus tentans cell line. -, inhibition; +, induction; -P, modulation of phosphorylation; Q, arrangement of microtubules changed; ®, transient induction; [], changed localization, patchwise occurrence. (1) Dinan et al., 1990; Spindler et al., 1993. (2) Rauch et al., in press. (3) LammerdingK/Sppel et al., 1994; Spindler-Barth et al., 1995; Wegener et al., 1996. (4) Fretz et al., in press. (5) Spindler-Barth et al., 1988, 1997; Spindler-Barth, 1991; Lammerding-K6ppel et al., 1994. 194 LAMMERDING-KOPPEL ET AL. REFERENCES Bryant, P.J. 1996. Cell proliferation control in Drosophila: flies are not worms. Bioessays, 18, 781-784. Buznikov, G.A., Shmukler, Y.B. and Lauder J.M. 1996. From oocyte to neuron: do neurotransmitters function in the same way throughout development? Cell. Mol. Biol., 16, 533-559. Dinan, L., Spindler-Barth, M. and Spindler, K.-D. 1990. Insect cell lines as tools for studying ecdysteroid action. J. Invertebrate Reprod. Dev., 18, 43-53. Drews, U. 1975. Cholinesterase in embryonic development. Progr. Histochem. Cytochem., 7, 1-52. Fretz, A., Lezzi, M. and Spindler, K.-D. Ecdysteroid dependent tissue differentiation and synthesis of cytoskeletal elements in an epithelial cell line from Chironomus tentans. In press. Hatt, P.J., Morni~re, M., Oberlander, H. and Porcheron, P. 1994. Roles for insulin and ecdysteroids in differentiation of an insect cell line of epidermal origin. In Vitro Cell Dev. Biol., 30A, 717-720. Hart, P.J., Liebon, C., Morni~re, M., Obeflander, H. and Porcheron, P. 1997. Activity of insulin growth factors and shrimp neurosecretory organ extracts on a lepidopteran cell line. Arch. Insect Biochem. Physiol., 34, 313-328. Hsu, S.-C. and deFranco, D.B. 1995. Selectivity of cell cycle regulation of glucocorticoid receptor function. J. Biol. Chem., 270, 3359-3364. Lammerding-K6ppel, M., Spindler-Barth, M. and Drews, U. 1994.20OH-ecdysone-induced morpbogenetic movements in a Chironomus cell line are accompanied by expression of an embryonic muscarinic system. Roux's Arch. Dev. Biol., 203,439-444. Milfin, M., Campuzano, S., and Garcia-Bellido, A. 1996. Cell cycling and patterned cell proliferation in the Drosophila wing during metamorphosis. Proc. Natl. Acad. Sci., 93, 11687-11692. Quack, S., Fretz, A., Spindler-Barth, M. and Spindler, K.-D. 1995. Receptor affinities and biological responses of nonsteroidal ecdysteroid agonists on the epithelial cell line from Chironomus tentans (Diptera: Chironomidae). Eur. J. Entomol., 92, 341-347. Rauch, P., Grebe, M., Elke, C., Spindler, K.-D. and Spindler-Barth, M. Ecdysteroid receptor and ultraspiracle from Chironomus tentans (Insecta) are phosphoproteins and regulated differently by molting hormone. Insect Biochem. Molec. Biol. In press. Segraves, W.A. 1994. Steroid response and other transcription factors in ecdysone response. Rec. Progr. Horm. Res., 49, 167-195. Spindler, K.-D., Quack, S. and Spindler-Barth, M. 1993. Insect cell lines as tools for insecticide screening. Trends Comp. Biocbem. Physiol., 1, 1045-1056. Spindler-Barth, M. 1991. Hormonal regulation of acetylcholine esterase in an epithelial cell line from Chironomus tentans. Z. Naturforsch., 46c, 1089-I093. Spindler-Barth, M., Schmidt, H., Drews, U. and Spindler, K.-D. 1988. Increase in activity of acetylcholinesterase by 20-OH-ecdysone in a Chironomus tentans cell line. Roux's Arch. Dev. Biol., 197, 366-369. Spindler-Barth, M., Junger, E., and Spindler, K.-D. 1992. Vesicle formation and ecdysteroid induced cellular differentiation in the epithelial cell line from Chironomus tentans. Tissue Cell, 24, 919-934. Spindler-Barth, M., Junger, E., Baumeister, R. and Spindler, K.-D. 1995. The epithelial cell line from Chironomus tentans: hormonal regulation of tissue differentiation and cuticle formation. Acres de Colloques, 18, 87-93. Spindler-Barth, M., Quack, S., Ranch, P. and Spindler, K.-D. 1997. Biological effects of muristerone A and turkesterone on the epithelial cell line from Chironomus tentans (Diptera: Chironomidae) and correlation with binding affinity to the ecdysteroid receptor. Eur. J. Entomol., 94, 161-166. Stevens, B., Alvarez, C.M., Bohman, R. and O'Connor, J.D. 1980. An ecdysteroid-induced alteration in the cell cycle of cultured Drosophila cells. Cell, 22, 675-682. Tata, J.R. 1994. Autoregulation and crossregulation of nuclear receptor genes. Trends Endocrinol. Metab., 5, 283-290. Wegener, S., Spindler-Barth, M. and Spindler, K.-D. 1996. A muscarinic acetylcholine receptor, present in the epithelial cell line from Chironomus tentans. Biol. Chem., 377, 819-824. Wegmann, I., Quack, S., Spindler, K.-D., Dorsch-H~isler, K., V6gtli, M. and Lezzi, M. 1995. Immunological studies on the developmental and chromosomal distribution of ecdysteroid receptor protein in Chironomus tentans. Arch. Insect Biochem. Physiol., 30, 95-114. Wyss, C. 1980. Loss of ecdysterone sensitivity of a Drosophila cell line after hybridization with embryonic cells. Exp. Cell Res., 125, 121-126. Wyss, C. 1982. Chironomus tentans epithelial cell lines sensitive to ecdysteroids, juvenile hormone, insulin and heat shock. Exp. Cell Res., 139, 309-319. Yao, T.P., Forman, B.M., Jiang, Z.Y., Cherbas, L., Chen, J.D., McKeown, M., Cherbas, P. and Evans, R.M. 1993. Functional ecdysone receptor is the product of EcR and Ultraspiracle. Nature, 366, 476-479.