EcR expression in the prothoracicotropic hormone-
producing neurosecretory cells of the Bombyx mori brain
An indication of the master cells of insect metamorphosis
Monwar Hossain
1
, Sakiko Shimizu
2
, Haruhiko Fujiwara
3
, Sho Sakurai
1,2
and Masafumi Iwami
1,2
1 Division of Life Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Japan
2 Division of Biological Science, Graduate School of Natural Science and Technology, Kanazawa University, Japan
3 Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan
The insect brain is the center of developmental control.
In the brain of the silkworm Bombyx mori, two pairs
of lateral neurosecretory cells (LNCs) produce the
prothoracicotropic hormone (PTTH) [1,2]. The peptide
PTTH stimulates the prothoracic glands to synthesize
and release ecdysone [3]. The active form of ecdysone,
20-hydroxyecdysone (20E), controls many physiologi-
cal and developmental processes of insect molting and
metamorphosis [4]. Beside PTTH, the brain produces
many neurosecretory hormones that orchestrate devel-
opmental processes. It is also the target organ of 20E,
effecting dynamic morphological changes and rear-
rangement of the neural network during processes such
as the formation of the optic lobe and mushroom body
differentiation during metamorphosis [5,6]. Hence, the

tors, the ecdysone receptor (EcR) and ultraspiracle (USP). Expression of
the specific isoforms EcR-A and EcR-B1 governs steroid-induced responses
in the developing cells of the silkworm Bombyx mori. Here, analysis of
EcR-A and EcR-B1 expression during larval-pupal development showed
that both genes were up-regulated by 20E in the B. mori brain. Whole-
mount in situ hybridization and immunohistochemistry revealed that
EcR-A and EcR-B1 mRNAs and proteins were exclusively located in two
pairs of lateral neurosecretory cells in the larval brain known as the pro-
thoracicotropic hormone (PTTH)- producing cells (PTPCs). In the pupal
brain, EcR-A and EcR-B1 expression was detected in tritocerebral cells and
optic lobe cells in addition to PTPCs. As PTTH controls ecdysone secre-
tion by the prothoracic gland, these results indicate that 20E-responsive
PTPCs are the master cells of insect metamorphosis.
Abbreviations
20E, 20-hydroxyecdysone; EcR, ecdysone receptor; LNC, lateral neurosecretory cell; MEF2, myocyte enhancer factor 2; OLC, optic lobe cell;
NaCl ⁄ P
i
⁄ Tween, phosphate buffered saline containing Tween; PTPC, PTTH-producing neurosecretory cell; PTTH, prothoracicotropic
hormone; TCC, tritocerebral cell; USP, ultraspiracle.
FEBS Journal 273 (2006) 3861–3868 ª 2006 The Authors Journal compilation ª 2006 FEBS 3861
20E. EcR-A expression regulates neuronal maturation,
while EcR-B1 expression controls neuronal regression
both in Drosophila melanogaster [14,15] and Manduca
sexta [16]. Mutational analysis of EcR isoforms in
Drosophila identified several different lethal phases,
including developmental arrest late in embryogenesis,
failure of pupariation [17,18], and disruption of neur-
onal remodeling [18]. Although the effect of ecdysone
on neurons of the central nervous system has been
extensively studied in Drosophila and Manduca, little

RT-PCR (Fig. 1C). Strong EcR-B1 expression was also
detected in the anterior part of the brain and weaker
expression was detected in the posterior section, indi-
cating that both genes exhibit a spatial-specific pattern
of expression. RpL32 expression did not differ between
the two parts of the brain.
Cell-specific expression of EcR isoforms as
revealed by in situ hybridization
The spatial specificity of EcR-A and EcR-B1 expres-
sion was also determined by whole-mount in situ
hybridization of larval and pupal brains using EcR iso-
form-specific probes. EcR-A expression was detected
exclusively in two pairs of lateral neurosecretory cells
(LNCs) in day-2 (Fig. 2A) and day-7 (Fig. 2C) fifth
instar larval brains. The location of the EcR-A-positive
cells was assumed to be the same as that of PTTH-
producing cells (PTPCs), as shown in Fig. 2E. To con-
firm this, we performed in situ hybridization using a
mixture of EcR-A and PTTH probes. The hybridiza-
tion signal was again detected exclusively in the two
pairs of LNCs (Fig. 2G), indicating that EcR-A and
PTTH mRNAs were colocalized in PTPCs of the lar-
val brain. EcR-B1 expression was also detected exclu-
sively in the same LNCs in day-2 (Fig. 2B) and day-7
(Fig. 2D) fifth instar larval brains, while EcR-B1 and
PTTH expression was shown to colocalize in PTPCs
of the larval brain (Fig. 2H).
In the pupal brain, EcR-A expression was observed
in PTPCs (Fig. 2I,L) and in several tritocerebral cells
(TCCs) (Fig. 2K) and optic lobe cells (OLCs) (Fig. 2J).

same results were obtained for EcR-B1 (Fig. 3A–C,
right), confirming that both isoforms are colocalized in
PTPCs in the brain.
In the pupal brain, EcR-A and EcR-B1 expression
was observed in TCCs and weakly in OLCs in addi-
tion to PTPCs (Fig. 3D,E, also Fig. 2I,J). It is note-
worthy that EcR-B1 fluorescence was strongest just
beneath the cell membrane of TCCs (Fig. 3F) and
was weaker in the axons, while EcR-A fluorescence
was more diffuse throughout the cytoplasm (Fig. 3D,
also Fig. 3A), indicating that the distribution of the
two isoforms differed slightly. EcR-A and EcR-B1
expression was also detected in the subesophagal gan-
glion (Fig. 3D,E).
Discussion
In the present study, we have shown for the first
time that EcR-A and EcR-B1 are up-regulated by 20E
AB
DC
EF
HG
I
J
K
L
Fig. 2. Localization of EcR-A, EcR-B1 and
PTTH mRNAs by whole-mount in situ
hybridization. EcR-A and EcR-B1 mRNA was
detected in day-2 (A,B) and day-7 (C,D) fifth
instar larval brains and day-2 pupal brains

immediately met by the consumption of a blood meal,
thus initiating development. No such phenomenon
exists in Bombyx larvae, and it is possible that the
difference in 20E-responsive cells between the two
insects reflects the different developmental systems.
Our study indicates that the expression level of EcR-
A is slightly higher than that of EcR-B1, as previously
observed during the neuronal maturation of Drosophila
and Manduca [16]. The developmental function of
EcR-A is distinct from those mediated by the EcR-B1
and EcR-B2 isoforms in Drosophila.AnEcR-A mutant
is arrested during early to mid-pupal development,
indicating that EcR-A is required for the formation of
the basic pupal body plan prior to the differentiation
of most adult structures [15]. By contrast, an EcR-B1
mutant is lethal at the first and second larval molts
[18] and fails to undergo pupariation [17].
PTPCs are the only cells to express EcR isoforms in
the Bombyx larval brain, and these cells also express
A
B
C
D
F
E
Fig. 3. Immunohistochemical localization of
EcR-A, EcR-B1, and PTTH. (A) Day-2 larval,
(B) day-7 larval and (C) day-2 pupal brains.
For fifth instar day-2 specimens, brains were
dissected 6 h after 20E injection. Anti-EcR-A,

expression in the PTPCs suggests that 20E modulates
PTTH expression through a feedback loop. Beside its
prothoracicotropic effect, PTTH may act as a growth
factor as it shares a common ancestor with the verteb-
rate growth factor superfamily peptides such as nerve
growth factor, transforming growth factor, and plate-
let-derived growth factor [21]. It also enhances the syn-
thesis of several short-lived proteins that mediate a
variety of extracellular signals [22,23]. Despite the first
demonstration of PTTH almost three decades ago in
LNCs in Manduca [24] and later in Bombyx [1,2],
the molecular mechanisms of PTTH production and
release are still at an early level of understanding,
although these are of critical importance in insect
developmental control [25].
Since the work of Truman [26], it has been believed
that PTTH release is controlled by a circadian clock in
the brain [27–29]. The close association between clock
cells and PTPCs in the brain protocerebral region is
seen in the three divergent genera Rhodnius, Dro-
sophila, and Bombyx, suggesting that there are routes
of communication between these two cell populations
[30,31]. The association with clock cells and the
responsiveness of PTPCs for ecdysteroidogenesis sug-
gests that 20E influences PTTH release from PTPCs
through neurons that provide input to clock cells
[31,32]. The PTTH titer in Bombyx larval hemolymph
is, however, not exclusively controlled by photoperiod
and ⁄ or circadian clock mechanisms [27,28], as protho-
racicostatic hormones have been shown to influence

expression in the brain is exclusive to the PTPCs until
pupation. This indicates that PTPCs are the master
cells during larval-pupal metamorphosis and that they
control ecdysteroidgenesis. At the pupal stage, the
number of cells expressing EcR isoforms increases to
include TCCs and OLCs. This unique expression pro-
file indicates the importance of EcR isoform expression
in TCCs and OLCs during larval-pupal metamorpho-
sis, although the roles of the individual isoforms in
these cells remain to be elucidated.
Experimental procedures
Animals and hormones
B. mori eggs of a racial hybrid, Kinshu · Showa were
obtained from Ueda Sanshu (Ueda, Japan), and larvae
were reared on an artificial diet (Silkmate II, Nihon Nou-
san Kogyo, Yokohama, Japan) under a 12 h light ⁄ 12 h
dark photoperiod at 25 ± 1 °C [38]. Ages were counted in
days, consisting of a photophase followed by a scotophase.
Fifth instar day-2 and day-7 larvae and day-2 pupae were
studied. 20E (Sigma, St. Louis, MO, USA) was dissolved in
ethanol and its concentration was determined spectrophoto-
metrically at 243 nm (e
EtOH
¼ 12 300). An aliquot of the
stock solution was evaporated and dissolved in insect Ring-
er’s solution (130 mm NaCl, 4.7 mm KCl, 1.9 mm CaCl
2
).
RNA isolation and semiquantitative RT-PCR
Total RNA was isolated from whole brains 2 h after the

subjected to 25 or 35 cycles of amplification in a thermo-
cycler (GeneAmp PCR System 9700, Applied Biosystems,
Foster City, CA, USA) using a thermal cycle (94 °C, 30 s;
60 °C, 30 s; 72 °C, 30 s). PCR products were separated on
a 1.5% (w ⁄ v) agarose gel and visualized by ethidium bro-
mide staining. There was no amplification without reverse
transcriptase even at 35 cycles of PCR (data not shown),
indicating the specificity of EcR-A and EcR-B1 mRNA
amplification.
In situ hybridization
Whole-mount in situ hybridization of brains was performed
as described previously [41]. After dissecting, brains were
washed with 10 mm phosphate buffered saline, pH 7.4
NaCl ⁄ P
i
⁄ 0.05% Tween20 and fixed in 85% (v ⁄ v) ethanol,
4% (w ⁄ v) formaldehyde, and 5% (v ⁄ v) acetic acid on ice
for 40 min. The brains were then incubated in a solution of
NaCl ⁄ P
i
containing 15% (w ⁄ v) sucrose at 4 °C for 15–20 h.
After washing with NaCl ⁄ Pi ⁄ 0.05% Tween20, the brains
were treated with proteinase K (0.05 mgÆmL
)1
in NaCl ⁄
Pi ⁄ 0.05% Tween20) at 37 °C for 40 min, and fixed again
with 3% (w ⁄ v) paraformaldehyde in NaCl ⁄ P
i
at room tem-
perature for 20 min. The brains were washed three times

methyl salicylate and observed with a microscope (BX-50F,
Olympus, Tokyo, Japan). Negative controls omitted the
labeled probes, and no signal was detected (data not
shown).
Anti-peptide serum for Bombyx EcR isoforms
An EcR-A specific peptide [H
2
N-GQVKAEPGVSHN
GHP(15–29)C-COOH] [13] (Accession no D87118) and an
EcR-B1 specific peptide [H
2
N-CPLPMPPTTPKSENES
MSSG(94–112)-COOH] [12] (Accession no D43943) were
synthesized, HPLC purified, and used to immunize rabbits
(Sawady Technology, Tokyo, Japan). Handling of rabbits
was performed according to regulation and guidelines of
the local authority. The anti-peptide serum titers for EcR-A
and EcR-B1, determined using an enzyme-linked immuno-
sorbent assay, were 4700 and 119 700, respectively.
Immunohistochemistry
Double-labeled fluorescent immunohistochemistry was used
to detect EcR-A, EcR-B1, and PTTH expression. Brains
were washed with NaCl ⁄ P
i
, fixed in Bouin’s solution over-
night, then washed with 70% (v ⁄ v) ethanol. The brains
were then soaked in 0.1% (w ⁄ v) sodium deoxycholate and
2% (v ⁄ v) Tween20 in NaCl ⁄ P
i
at 4 °C for 4 days to facili-

18380040) from the Japan Society for the Promotion
of Science.
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EcR expression in PTTH cells of Bombyx mori brain M. Hossain et al.
3868 FEBS Journal 273 (2006) 3861–3868 ª 2006 The Authors Journal compilation ª 2006 FEBS