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.
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