Báo cáo khoa học: Myocyte enhancer factor 2 (MEF2) is a key modulator of the expression of the prothoracicotropic hormone gene in the silkworm, Bombyx mori ppt

Myocyte enhancer factor 2 (MEF2) is a key modulator
of the expression of the prothoracicotropic hormone gene
in the silkworm, Bombyx mori
Kunihiro Shiomi
1
, Yoshihiro Fujiwara
1
, Tsutomu Atsumi
1
, Zenta Kajiura
1
, Masao Nakagaki
1
,
Yoshiaki Tanaka
2
, Akira Mizoguchi
3
, Toshinobu Yaginuma
4
and Okitsugu Yamashita
4,5
1 Faculty of Textile Science and Technology, Shinshu University, Nagano, Japan
2 National Institute of Agrobiological Sciences (NIAS), Ibaraki, Japan
3 Graduate School of Science, Nagoya University, Aichi, Japan
4 Graduate School of Bioagricultural Sciences, Nagoya University, Aichi, Japan
5 Chubu University, Aichi, Japan
Organisms have adapted to seasonal fluctuations by
evolving internal clocks and neuroendocrine systems
to anticipate variations in living conditions [1]. In
insects, prothoracicotropic hormone (PTTH) secretion

appears to be triggered by a particular set of environ-
mental signals, including the photoperiod and tem-
perature [2–4]. PTTH stimulates the prothoracic
glands to synthesize and release ecdysone, the steroid
necessary for molting, metamorphosis, and the termin-
ation of pupal diapause [2–4]. PTTH was first purified
and sequenced from the silkworm, Bombyx mori [5,6].
The PTTH gene is constantly expressed during larval–
pupal development [7], and the peptide is produced
exclusively in two pairs of lateral PTTH-producing
neurosecretory cells (PTPCs) in the brain [8]. From
there it is transported via axons to the corpora allata
and then released into the hemolymph. The PTTH
titer in the hemolymph has been shown to correlate
Keywords
baculovirus; Bombyx mori; MEF2;
metamorphosis and diapause; PTTH
Correspondence
K. Shiomi, Faculty of Textile Science and
Technology, Shinshu University, Ueda,
Nagano, 386-8567, Japan
Fax: +81 268 21 5331
Tel: +81 268 21 5338
E-mail:
Database
The sequences reported in this paper have
been deposited in the DDBJ database under
Accession no. AB121093.
(Received 14 April 2005, revised 24 May
2005, accepted 31 May 2005)

doi:10.1111/j.1742-4658.2005.04799.x
Prothoracicotropic hormone (PTTH) plays a central role in controlling
molting, metamorphosis, and diapause termination in insects by stimulating
the prothoracic glands to synthesize and release the molting hormone,
ecdysone. Using Autographa californica nucleopolyhedrovirus (AcNPV)-
mediated transient gene transfer into the central nervous sytem (CNS) of
the silkworm, Bombyx mori, we identified two cis-regulatory elements that
participate in the decision and the enhancement of PTTH gene expression
in PTTH-producing neurosecretory cells (PTPCs). The cis-element media-
ting the enhancement of PTTH gene expression binds the transcription fac-
tor Bombyx myocyte enhancer factor 2 (BmMEF2). The BmMEF2 gene
was expressed in various tissues including the CNS. In brain, the BmMEF2
gene was expressed at elevated levels in two types of lateral neurosecretory
cells, namely PTPCs and corazonin-like immunoreactive lateral neurosecre-
tory cells. Overexpression of BmMEF2 cDNA caused an increase in the
transcription of PTTH. Therefore, BmMEF2 appears to be particularly
important in the brain where it is responsible for the differentiation of lat-
eral neurosecretory cells, including the enhancement of PTTH gene expres-
sion. This is the first report to identify a target gene of MEF2 in the
invertebrate nervous system.
Abbreviations
AcNPV, Autographa californica nucleopolyhedrovirus; BmMEF2, Bombyx mori myocyte enhancer factor 2; CLI-LNCs, corazonin-like
immunoreactive lateral neurosecretory cells; CNS, central nervous system; DIG, digoxigenin; EGFP, enhanced green fluorescence protein;
MADS box, MCM1-Agamous-Deficiens-Serum response factor box; PTPCs, PTTH-producing neurosecretory cells; PTTH, prothoracicotropic
hormone; SG, subesophageal ganglion.
FEBS Journal 272 (2005) 3853–3862 ª 2005 FEBS 3853
closely with the ecdysteroid titer [3,9]. Fluctuations of
PTTH titer in hemolymph consequentially act as a
pacemaker in the neuroendocrine regulation of develo-
pment by varying the secretion of ecdysone. The tim-

ing of the increase in hemolymph PTTH titer on the
day of wandering is photoperiodically controlled in
B. mori [3]. In addition, in larvae of Heliothis vires-
cens, expression of the PTTH gene declines sharply at
the onset of larval wandering behavior and remains
low during pupal diapause [10]. Thus, analysis of the
molecular mechanisms controlling PTTH secretion in
PTPCs is important for understanding the termination
of pupal diapause as well as the induction of molting
and metamorphosis.
In the current study, we developed a convenient sys-
tem for transiently transferring genes into the central
nervous system (CNS) of B. mori using the recombin-
ant baculovirus, AcNPV [11]. Using this system, we
have been able to preferentially express the enhanced
green fluorescence protein (EGFP) reporter gene under
control of the PTTH promoter in PTPCs [11]. We
used this system to investigate the molecular mecha-
nisms controlling PTTH secretion by PTPCs. In the
present report, we focused on the regulation of PTTH
gene expression and found that the Bombyx myocyte
enhancer factor 2 (BmMEF2) binds to the PTTH pro-
moter and enhances its gene expression. Thus, the
PTTH gene was identified as the first known target
gene of MEF2 in the invertebrate nervous system.
BmMEF2 appears to be particularly important in the
brain where it causes the differentiation of lateral neuro-
secretory cells by enhancing PTTH gene expression.
Results
Expression of the PTTH reporter gene is

regulated by two cis-elements
To determine the cis-elements participating in the regu-
lation of PTTH gene expression, we performed repor-
ter gene analysis using an AcNPV-mediated gene
transfer system. We first examined whether the repor-
ter gene construct containing EGFP under control
of nucleotides )879 to +52 of the PTTH promoter
(v[PT ⁄ EGFP]) [11] is expressed in the somata and
neurites of PTPCs (Fig. 1). The fluorescence was
localized within two pairs of lateral cells in the proto-
cerebrum, and a faint signal was found in many cells
throughout the brain lobes and at the midline in the
subesophageal ganglion (SG) (Fig. 1A). The axons
emanating from the somata of the two pairs of lateral
cells extend towards the pars intercerebral with some
arborization (Fig. 1A, box and Fig. 1B), run contralat-
eral after crossing the pars intercerebral (Fig. 1A,B,
arrow), and then project into the corpora allata with
varicosites (Fig. 1F). Immunohistochemical staining
with an anti-PTTH IgG to visualize endogenous PTTH
produced by PTPCs identified two pairs of lateral cells
in the protocerebrum (Fig. 1C) [8]. Merging Cy3 (anti-
PTTH) with EGFP (v[PT ⁄ EGFP]) signals in the somata
and axons of the cells (Fig. 1D,E) revealed many gran-
ules on the cell surface, although most of the EGFP sig-
nal was localized preferentially in the nucleus (Fig. 1E).
Furthermore, the Cy3 signals in the PTPCs projected
into the corpus allatum–corpus cardiacum complex
where most of the signal overlapped with the EGFP
signal (Fig. 1F). Thus, the neurosecretory cells in

the brain of Bombyx expressing v[PT ⁄ EGFP]-derived
EGFP corresponded to PTPCs. The results also sug-
gest that the sequence of the PTTH promoter from
nucleotides )879 to +52 contains cis-regulatory ele-
ments that drive PTTH gene expression in PTPCs.
We constructed six recombinant AcNPVs carrying
different upstream regions of the PTTH gene fused
with the EGFP reporter gene. EGFP fluorescence was
observed in PTPCs (Fig. 1G–M). We also measured
the fluorescence intensity in somata and compared
it with the intensity of the recombinant AcNPV
(v[PT ⁄ EGFP]) carrying nucleotides )879 to +52 of
the PTTH promoter (n ¼ 22) (Fig. 1R). Progressive
deletion of the 5¢-upstream region, either from nucleo-
tides )208 to +52 or from )180 to +52, had no signi-
ficant effect on EGFP expression (97.6 ± 4.0%, n ¼
21 and 95.5 ± 7.7%, n ¼ 25, respectively; Fig. 1G–I).
However, recombinant AcNPVs carrying nucleotides
)167 to +52 and or )119 to +52 of the PTTH pro-
moter caused an abrupt decrease in the expression
of EGFP in the PTPCs (49.7 ± 19.0%, n ¼ 46 and
44.3 ± 16.9%, n ¼ 42, respectively; Fig. 1J,K). Using
a recombinant AcNPV carrying nucleotides )105 to
+52 of the PTTH promoter, EGFP expression was
faint, and no fluorescence signal was observed in some
pupa (9.9 ± 9.8%, n ¼ 37; Fig. 1L,L¢). No expression
was observed when nucleotides )60 to +52 of the
PTTH promoter were used (2.1 ± 4.1%, n ¼ 9;
Fig. 1M), although faint signals on small cells were
still detected in the lateral brain. Injection with recom-

binant AcNPVs carrying nucleotides )879, )208,
)180, )167, )119, )105, or )60 to +52 of the PTTH
promoter resulted in hemolymph virus titers of 9.01,
7.42, 9.92, 8.64, 9.04, 9.15, and 10.15 · 10
6
pfuÆmL
)1
,
indicating that there was no significant difference in
the ability of the various viral constructs to infect the
pupae.
In addition to injection of pupae with AcNPVs, we
also examined the effect of injections into day 0 of
Enhancement of PTTH gene expression by MEF2 K. Shiomi et al.
3854 FEBS Journal 272 (2005) 3853–3862 ª 2005 FEBS
fifth instar larvae (Fig. 1N–Q). As in pupal brain,
fluorescence due to injection of v[PT ⁄ EGFP] was
observed in two pairs of lateral neurosecretory cells
(Fig. 1N) that corresponded to the PTPCs (data not
shown). In the larval brain infected with recombinant
AcNPVs carrying nucleotides )180 to +52, )167 to
+52, or )105 to +52 of the PTTH promoter, the
relative fluorescence intensities of EGFP were 82.3 ±
47.2% (n ¼ 35) (Fig. 1O), 40.1 ± 15.7% (n ¼ 20)
(Fig. 1P), and 5.7 ± 5.1% (n ¼ 20) (Fig. 1Q), respect-
ively. Injection with recombinant AcNPVs carrying nu-
cleotides )879, )180, )167, or )105 to +52 of the
PTTH promoter resulted in hemolymph virus titers of
3.52, 5.01, 2.92 and 4.27 · 10
8

pfuÆmL
)1
, indicating
that there was no significant difference in the ability of
the different viral constructs to infect the larvae. Thus,
using EGFP reporter gene analysis and serial deletion
of the PTTH promoter, we identified two potential cis-
regulatory elements: (a) a 61 bp sequence from nucleo-
tide )180 to )119 that participates in the enhancement
of PTTH gene expression, and (b) a 15 bp sequence
from nucleotide )119 to )105 that helps direct the
expression of the PTTH gene expression in PTPCs. It
appears that these two cis-elements are functionally
conserved during larval–pupal development.
The MEF2-binding sequence is important for
enhancing PTTH gene expression
To identify the trans-activating factors that enhance
PTTH gene expression, we searched the 61 bp
sequence from nucleotide )180 to )119 of the PTTH
gene for transcription factor-binding sites using mat-
inspector ( As shown in
Fig. 2A, we found that the DNA sequence bound by
MEF2, C ⁄ TTA(A ⁄ T)
4
TAG ⁄ A [12], is conserved at the
5¢-upstream region from nucleotides )180 to )151 of
the PTTH gene (CACAATGGTT
CTATTTTAAG
GATTTATCAC; MEF2 binding consensus underlined;
Fig. 2A, wt).

A gel-mobility shift assay using a 30-bp double-
stranded oligonucleotide encoding nucleotides )180 to
)151 of the PTTH promoter (Fig. 2A, wt) as a probe
showed a shifted band (Fig. 2B, lane 1) that was pro-
gressively lost upon incubation with increasing concen-
trations of unlabeled wt oligonucleotide (Fig. 2B, lanes
2–4). We further synthesized three double-stranded
oligonucleotides as competitors to analyze the
sequence specificity of the protein bound to the wt
oligonucleotide. In oligonucleotide M1, the MEF2
consensus binding sequence was disrupted by mutation
A
OL
B

r
S

G
CA CA


O

L
J
I
K
FGH
D

C
B
E
ML
L'
R
-879
-208
-180
-167
-119
-105
-60
0 25 50 75 100
Relative fluorescence intensity (%)
C

o

n

s

t

r

u

c

t

s
G
H
I
J
K
L
M
P
Q
N
O
Fig. 1. Identification of cis-regulatory
elements controlling PTTH gene expression
in brain PTPCs of B. mori using AcNPV-
mediated reporter gene analysis. Fluores-
cence microscopy was used to visualize
EGFP expression in the brain–SG complexes
of larvae (N–Q) and pupae (A–M) injected
with recombinant AcNPVs expressing
v[PT ⁄ EGFP] carrying nucleotides )879 to
+52 (A–G and N), )208 to +52 (H), )180 to
+52 (I, O), )167 to +52 (J, P), )119 to +52
(K), )105 to +52 (L, L¢,Q),or)60 to +52 (M)
of the PTTH gene. The axon emanating from
the somata (light blue arrowheads and
enlarged image shown in E) runs towards the

midline of the brain with some arborization
(boxed area in A), contralateral after crossing
the midline (arrow in A–D), and then projects
to the corpus allatum (F). Magnified images
(B–E and G–Q) show the somata and axon
indicated by the box in (A). In B-F, the PTPCs
in a v[PT ⁄ EGFP]-injected pupae were exam-
ined by immunohistochemistry with an
anti-PTTH mAb (magenta). The EGFP fluores-
cence was visualized by the green color. The
relative fluorescence intensity of the PTPCs
are shown as the percentage compared with
v[PT ⁄ EGFP]-injected pupa (R). Br, brain; SG,
subesophageal ganglion; CA, corpus allatum;
OL, optic lobe. Scale bar ¼ 50 lm.
K. Shiomi et al. Enhancement of PTTH gene expression by MEF2
FEBS Journal 272 (2005) 3853–3862 ª 2005 FEBS 3855
of 2 bp (Fig. 2A, M1). M2 contained the sequences of
optimal targets for MEF2 expressed in mouse brain
[12] (Fig. 2A, M2). In M3, 3 bp were mutated, but
they are not within the MEF2 consensus binding
sequence (Fig. 2A, M3). Even at a 100-fold excess, M1
was unable to compete the binding of protein to wt
(Fig. 2B, lane 5). However, M2 eliminated protein
binding to wt (Fig. 2B, lane 6); in fact, competition by
M2 was stronger than with unlabeled wt (Fig. 2B;
lanes 2–4). The shifted band also decreased in the pres-
ence of M3 (Fig. 2B, lane 7) or anti-BmMEF2
(MADS) IgG (Fig. 2B, lane 10), but nonimmune
serum had no effect (Fig. 2B, lane 9). Thus, we found

that a protein in Bombyx brain bound to the MEF2
consensus binding sequence, and its binding was pre-
vented by an antiserum that recognizes the MADS
box of BmMEF2.
We examined this further using recombinant
AcNPVs carrying nucleotides )180 to +52 of the
PTTH promoter and the M1, M2,orM3 sequence
(Fig. 2A,C). Based on fluorescence intensity, the M3
virus was as effective (92.7 ± 5.4%, n ¼ 18; Fig. 2C,
panel M3) at mediating EGFP expression as the wt
virus (Fig. 2C, panel wt). The M2 virus resulted in an
enhanced level of fluorescence (113.3 ± 10.1%, n ¼
18; Fig. 2C, panel M2), and faint EGFP expression
was observed with the M1 virus, which contains a
disruption of the MEF2 binding consensus (36.4 ±
8.4%, n ¼ 18), although EGFP fluorescence was never
completely eliminated by this construct (Fig. 2C, panel
M1). Injection with recombinant AcNPVs wt, M1,
M2, and M3 resulted in hemolymph virus titers of
8.96, 7.35, 9.68, and 6.66 · 10
6
pfuÆmL
)1
, respectively,
indicating that there was not a significant difference in
the ability of the different virus constructs to infect the
pupae. Thus, the expression of EGFP was altered by
mutation of the MEF2 consensus binding sequence in
the PTTH promoter, a region important for enhancing
reporter gene expression. These findings suggest that

the Bombyx MEF2 homolog binds to the MEF2
consensus binding sequence in the PTTH promoter,
enhancing PTTH gene expression.
Cloning of the Bombyx MEF2 (BmMEF2) cDNA
We next cloned the MEF2 cDNA from the brain–SG
complex in Bombyx using a PCR-based strategy with
degenerate primers corresponding to the MADS-box
and the MEF2 domain [13], regions that are highly
conserved across a variety of organisms. A 2716-bp
sequence containing the 5¢- and 3¢-untranslated regions
of MEF2 (Accession no. AB121093) was obtained by
RT-PCR and rapid amplification of cDNA ends. The
open reading frame was from nucleotides +748 to
A
C
B
Fig. 2. Mutational analysis of the MEF2 consensus sequence (A) by gel mobility shift assay (B) and reporter gene analysis (C). The MEF2
consensus binding sequence in mouse brain [12] is boxed, and the 10 bp MEF2 core binding sequence is shown in capital letters. In the gel
mobility shift assay (B), the double-stranded oligonucleotide encoding from )180 to )151 of the PTTH gene (wt) was use as a probe. Oligo-
nucleotides M1, M2, and M3 were used as competitor DNAs. The mutated nucleotides are shown in bold. NS, normal rabbit serum; Ab,
anti-BmMEF2 (MADS). The shifted band is indicated by an arrow. Reporter gene expression was performed using a recombinant AcNPV car-
rying nucleotides )180 to +52 of the PTTH promoter and the nonmutated sequence (wt) or the M1, M2, or M3 mutant sequences. The
somata of PTPCs are indicated by light blue arrowheads.
Enhancement of PTTH gene expression by MEF2 K. Shiomi et al.
3856 FEBS Journal 272 (2005) 3853–3862 ª 2005 FEBS
+1965 and encoded a predicted 404-amino acid pro-
tein (Fig. 3A). A MADS box and an adjacent MEF2
domain are encoded within an 86-amino acid N-ter-
minal sequence (Fig. 3A). These two regions are highly
conserved in MEF2s from various organisms (Fig. 3B).

The Bombyx sequence is most similar to that of Dro-
sophila melanogaster (D-MEF2), with 96% amino acid
sequence identity in the MADS box and MEF2
domain (Fig. 3B).
Developmental expression of BmMEF2
We examined the developmental expression of
BmMEF2 in various tissues during embryonic and
postembryonic development by RT-PCR. BmMEF2
mRNA was first detected on day 3 after oviposition
(Fig. 4A, lane 2) and was detected thereafter through-
out embryogenesis, although the signal intensity of the
hybridized band decreased on day 9 after oviposition
(Fig. 4A, lane 4). During postembryonic development,
BmMEF2 mRNA was detected in various tissues con-
taining the brain–SG complex (Fig. 4A, lanes 5–15).
Intense signals were detected in the mixture of integu-
ment and muscle at both larval and pupal stages
(Fig. 4A, lanes 9 and 15) as well as in the fat body at
the pupal stage (Fig. 4A, lane 12). The PTTH mRNA
was exclusively expressed in the brain–SG complex
during postembryonic development (Fig. 4A, lanes 20–
30). During embryonic development, hybridized signals
were detected from day 3 (Fig. 4A, lane 17), which
corresponded to BmMEF2 expression (Fig. 4A, lane
2), although the signals were faint compared with
those in larval and pupal brain–SG complexes.
Next, we specifically examined the distribution of
BmMEF2 mRNA in the CNS by RT-PCR (Fig. 4B).
Although PTTH mRNA was detected exclusively in
brain (Fig. 4B, lane 1), the BmMEF2 mRNA was

detected in the SG and the first thoracic ganglion (T1)
as well as in the brain (Fig. 4B, lanes 1–3).
Furthermore, we determined the localization of
BmMEF2 mRNA in brain by whole-mount in situ
hybridization (Fig. 4C–I). Using an antisense
BmMEF2 RNA as a probe, we observed hybridization
throughout the brain, but it was particularly concen-
trated in cells within the lateral region of the protocer-
ebrum (Fig. 4C, blue box) and at the periphery of
the tritocerebrum (Fig. 4C, red box). Signals were not
detected when the sense strand RNA was used as a
probe (Fig. 4D). In the tritocerebrum, there were
intense hybridization signals that were reproducibly
detected in  20 cells in each hemisphere (Fig. 4E). In
contrast, in the lateral protocerebrum, the hybridiza-
tion signals were relatively weak, and different num-
bers of positive cells were observed among the 240
specimens (190 specimens with no positive cells, 26
with one positive cell, 12 with two positive cells, 9 with
three positive cells, and 4 with four positive cells).
Thus, in many specimens, hybridized signals in positive
large cells of the lateral brain were similar to levels of
neighboring cells.
To identify the lateral cells, we performed immuno-
histochemistry with the anti-PTTH IgG after in situ
hybridization. In some specimens, there were two
A
B
Fig. 3. Deduced amino acid sequence of the Bombyx MEF2 (A).
The MADS box and the MEF2 domain are shown in red and blue,

respectively. Alignment of the MADS box and the MEF2 domain of
BmMEF2 with that of (abbreviations and Accessions nos shown in
parentheses) Mus musculus MEF2A (mMef2a; U30823), Xenopus
laevis Mef2a (xMef2a; BC046368), Homo sapiens MEF2A
(hMEF2A; BC013437), Gallus gallus MEF2A (cMef2a; AJ010072),
Danio rerio mef2a (zMef2a; BC044337), Cyprinus carpio MEF2A
(CcMEF2A; AB012884), Caenorhabditis elegans mef-2 (Cemef-2;
U36199), Podocoryne carnea Mef2 (PcMef2; AJ428495), Coturnix
coturnix japonica qMEF2D (qMEF2D; AJ002238), Rattus norvegicus
MEF2D (rMEF2D; AJ005425), Halocynthia roretzi MEF2 (As-MEF2;
D49970), and Drosophila melanogaster D-MEF2 (D-MEF2; U07422)
(B). Identical amino acids are indicated with *.
K. Shiomi et al. Enhancement of PTTH gene expression by MEF2
FEBS Journal 272 (2005) 3853–3862 ª 2005 FEBS 3857
lateral cells showing BmMEF2 mRNA expression
(Fig. 4F) that also were stained with anti-PTTH
IgG (Fig. 4G). Moreover, in a few specimens, we
found that a hybridization signal in a lateral cell
corresponded to anticorazonin-immunoreactive cells
(Fig. 4H,I), although four corazonin-like immuno-
reactive lateral neurosecretory cells (CLI-LNCs) were
found in the lateral region of the brain in each
hemisphere [14].
Regulation of PTTH gene expression by controlling
BmMEF2 expression
To investigate whether the expression of BmMEF2
affects PTTH gene expression, we constructed two
recombinant AcNPVs, v[PT ⁄ MEFs] and v[PT ⁄ MEFi],
which were designed to overexpress and silence
BmMEF2 mRNA under control of the PTTH promo-

ter, respectively. When injected at 10
2
pfu per pupa,
the virus titers in hemolymph for v[PT ⁄ EGFP],
v[PT ⁄ MEFs], and v[PT ⁄ MEFi] were 5.12, 5.37, and
4.97 · 10
6
pfuÆmL
)1
, respectively, and when injected
at 10
6
pfu per pupa, the virus titers were 1.98, 1.18,
and 1.57 · 10
7
pfuÆmL
)1
. Therefore, we concluded that
there was no significant difference in the ability of the
different virus constructs to infect the pupae. Using
RT-PCR, we first investigated the effect of infection
with AcNPV on the amounts of mRNAs transcribed
from the BmMEF2, PTTH, and actin A3 genes. When
v[PT ⁄ EGFP] was injected at 10
2
pfu per pupa, we
could clearly detect the BmMEF2 mRNA, and we
could also detect it in noninjected pupae (Fig. 5A,
lanes 1, 2). However, when v[PT ⁄ EGFP] was injected
at 10

6
pfu per pupa, there was a slight decrease in the
amount of the BmMEF2 and PTTH mRNA (Fig. 5A,
lanes 3 and 10). Also, there were no changes in the
actin A3 mRNA (Fig. 5A, lanes 15, 16, and 17). These
results suggest that the AcNPV infection causes a
decrease in the amount of both BmMEF2 and PTTH
mRNA.
When v[PT ⁄ MEFs] was injected, there was a higher
level of BmMEF2 mRNA than in pupae that were not
injected or that were injected with v[PT ⁄ EGFP]
(Fig. 5A, lanes 4 and 5). Injection of v[PT ⁄ MEFi] at
both 10
2
and 10
6
pfu per pupa caused a large reduc-
tion of the BmMEF2 mRNA compared with non-
injected and v[PT ⁄ EGFP]-injected pupa (Fig. 5A, lanes
6 and 7). Thus, the two recombinant AcNPVs,
v[PT ⁄ MEFs] and v[PT ⁄ MEFi], were able to induce
overexpression and suppression of the BmMEF2 gene,
respectively. In addition, the amount of PTTH mRNA
was also increased by injection with v[PT ⁄ MEFs]
(Fig. 5A, lanes 11 and 12). However, v[PT ⁄ MEFi] did
not cause elimination of the PTTH mRNA (Fig. 5A,
lanes 13 and 14). Thus, BmMEF2 activated but was
not essential for PTTH gene expression.
F
G

I
H
BC D
E
ActA3
MEF2
Br SG T1
lane: 1 2 3
PTTH
A
2h 3d 5d 9d BS MG FB SL IM BS MG FB OV TS IM
MEF2
lane: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
ActA3
PTTH
lane:1617181920 21222324252627 282930
lane: 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
Fig. 4. Developmental profiles of expression of BmMEF2 and PTTH
genes. RT-PCR and Southern blot analysis (A, B) were performed
during embryogenesis 2 h (2 h) and 3 (3d), 5 (5d), and 9 (9d) days
after oviposition (Em) and on day 4 in fifth instar larvae (LV4) as
well as on day 3 in pupae (P3). BS, brain–subesophageal ganglion
complex; MG, midgut; FB, fat body; SL, silk gland; IM, integument
and muscle; OV, ovary; TS, testis; Br, brain; SG, subesophageal
ganglion; and T1, first thoracic ganglion. Whole-mount in situ
hybridization was performed in pupal brain by using antisense (C,
E–I) and sense (D) RNA of the BmMEF2 gene as probes. Magnified
images of the periphery of the tritocerebrum (E) and lateral brain
(F–I) are shown by the boxes in red and blue, respectively (C). The
hybridized signals in lateral brain (F, H) were examined by immu-

nohistochemistry with a monoclonal anti-PTTH IgG (magenta) (G) or
an anti-corazonin IgG (green) (I). Scale bar ¼ 100 lm.
PT/EGFP PT/MEFiPT/MEFs
C
10
2
10
6
10
2
10
6
10
2
10
6
(PFU/pupa)
lane: 1 2 3 4 5 6 7
MEF2
lane: 15 16 17 18 19 20 21
PTTH
ActA3
lane: 8 9 10 11 12 13 14
Fig. 5. Effect of PTTH gene expression on the overexpression and
silencing of the BmMEF2 gene. RT-PCR was performed on pupal
brain injected each of three recombinant AcNPVs (v[PT ⁄ EGFP],
v[PT ⁄ MEFs] and v[PT ⁄ MEFi]) at 10
2
and 10
6

pfu per pupa as well
as noninjected pupal brain (c). Levels of BmMEF2 (lanes 1–7),
PTTH (lanes 8–14), and ActinA3 (lanes 15–21) mRNAs were exam-
ined.
Enhancement of PTTH gene expression by MEF2 K. Shiomi et al.
3858 FEBS Journal 272 (2005) 3853–3862 ª 2005 FEBS
Discussion
We previously developed a system using AcNPV for
transient gene transfer into the CNS of the silkworm,
B. mori [11]. This system allows reporter gene analysis
of many constructs, enabling the identification of the
cis-elements in vivo. Furthermore, the system is highly
reproducible and can be set up within 2 weeks of con-
struction of the recombinant plasmids. In this study,
we used this system to identify the cis-elements and a
transcription factor responsible for expression of the
PTTH gene in vivo.
Within the PTTH promoter, we identified two
cis-regulatory elements participating in (a) the decision
to express the PTTH gene and (b) the enhancement of
PTTH gene expression. Our results indicate that the
5¢-upstream region from nucleotides )119 to )105 of
the PTTH gene participates in the decision to express
the PTTH gene (Fig. 1). We analyzed the cis-regula-
tory elements and a trans-activating factor participa-
ting in the decision to express the PTTH gene.
The 5¢-upstream region of the PTTH gene from
)180 to )151 is similar to the MEF2 consensus bind-
ing sequence of a variety of organisms. MEF2 belongs
to the family of MADS box transcription factors,

which bind to DNA as homo- and heterodimers
through the consensus MEF2 binding sequence,
C ⁄ TTA(A ⁄ T)
4
TAG ⁄ A [12]. This sequence is found in
the upstream regions of numerous genes including
muscle-specific genes, and plays a critical role in the
differentiation of cells during the development of
multicellular organisms [15]. There are four isoforms
(A–D) of mammalian MEF2, and they have high
homology within the 56-amino-acid MADS box at
their N-termini and within an adjacent 29-amino-acid
region referred to as the MEF2 domain. The MADS
box is essential for DNA binding and dimerization,
and the MEF2 domain plays an important role in
DNA binding affinity as well as an indirect role in
dimerization. The C-terminal portion of MEF2C is
required for its transcriptional activation [13].
The N-terminal 86 amino acids of BmMEF2 are
highly conserved and include a MADS box and a
MEF2 domain. We found that BmMEF2 binds to the
consensus sequence via its MADS box and can acti-
vate transcription of the target gene in B. mori as
well as MADS box-containing genes in various
other organisms. Furthermore, the BmMEF2 gene is
expressed in various tissues containing muscle and
neural tissues in Bombyx as well as in D. melanogaster
and various vertebrates. Consequently, correlation
between the structure and gene expression profiles sug-
gests that the BmMEF2 is a structural and functional

analog of MEF2 proteins in various organisms. Fur-
thermore, it has been speculated that BmMEF2 is
responsible for the regulation of fundamental cellular
processes in various tissues.
In this study, we demonstrated that the MEF2 bind-
ing sequences in the PTTH promoter enhance expres-
sion of the EGFP reporter gene in PTPCs and are
important for binding of the BmMEF2 protein. Fur-
thermore, overexpression of the BmMEF2 gene can
induce PTTH gene expression. Thus, it appears that
BmMEF2 plays a role in the enhancement of PTTH
gene expression in PTPCs. A single MEF2 gene,
d-mef2, has been identified in D. melanogaster, and the
isoforms of the D-MEF2 protein act as functional ana-
logs of the vertebrate forms that participate in muscle
differentiation [16,17]. Furthermore, D-MEF2 protein
is expressed in Kenyon cells in the mushroom bodies
of larval and adult brains, suggesting that these pro-
teins are responsible for the differentiation of the Ken-
yon cells and for morphogenesis of the mushroom
body learning center [18]. However, the target genes
for D-MEF2 have not been identified, and MEF2
functions have not been determined in the insect ner-
vous system. Thus, our findings are the first identifica-
tion of a gene that is a target of MEF2 in the
invertebrate nervous system.
Expression of the PTTH gene was first detected on
day 3 of embryogenesis. In addition, Adachi-Yamada
et al. [7] showed that it is constantly expressed during
larval–pupal development. The correlation between

PTTH and BmMEF2 gene expression suggests that
BmMEF2 activates PTTH expression throughout
embryonic and postembryonic development.
We found that the BmMEF2 gene is preferentially
expressed not only in PTPCs, but also in CLI-LNCs.
In Manduca sexta [19] as well as in Bombyx [14], CLI-
LNCs are identified as type Ia
1
neurosecretory cells.
These cells coexpress PERIOD and various peptides,
such as FMRFamide, and leu-enkephalin [20,21].
Furthermore, the genes of Antheraea pernyi, timeless,
and period are also expressed exclusively in four pairs
of cells in protocerebral lateral neurosecretory cells,
which are likely type Ia
1
neurosecretory cells. The close
anatomical localization between PTPCs and CLI-
LNCs suggests that there are routes of communication
between these two cell populations that may be
important for the circadian control of PTTH release
[22]. Although it is not known whether the two types
of neurosecretory cells communicate, the BmMEF2
gene may be activated via a common specialized mech-
anism in both PTPCs and CLI-LNCs and may thereby
participate in the terminal differentiation processes of
these lateral neurosecretory cells.
K. Shiomi et al. Enhancement of PTTH gene expression by MEF2
FEBS Journal 272 (2005) 3853–3862 ª 2005 FEBS 3859
Aizono and Shirai suggested that muscarinic acetyl-

choline receptor-induced signal transduction was
involved in the control of PTTH release in B. mori
[23]. Activation of phospholipase C and the subsequent
activation of both protein kinase C and calmodulin-
dependent kinase were essential in this signaling path-
way. Furthermore, MEF2 is known to act as an
endpoint for growth factor signaling pathways [24].
Although we identified BmMEF2 as a factor that
enhances PTTH gene expression, BmMEF2 may parti-
cipate in several other cellular processes that regulate
PTTH secretion through signaling pathways. Thus,
it will be important to further investigate the signal
transduction pathway by which extracellular signals
regulate insect functions including molting, metamor-
phosis, and diapause.
Experimental procedures
Animals
The polyvoltine strain, N4, of B. mori was used throughout
these experiments. Eggs were incubated at 25 °C under con-
tinuous darkness. Larvae were reared on an artificial diet
(Silkmate-2M, Nosan Co., Yokohama, Japan) at 25–27 °C
under a 12 h light ⁄ 12 h dark cycle. Larvae and pupae used
in the experiments were collected within 1 h after each
ecdysis (referred to as day 0) to synchronize their subse-
quent development. Pupae were kept at 25 °C to allow
adult development. Injection of recombinant AcNPV was
performed according to Shiomi et al. [11].
Preparation of recombinant AcNPV
Recombinant AcNPVs were prepared according to the
manufacturer’s instructions (Invitrogen, Carlsbad, CA,

USA) and Shiomi et al. [11]. For reporter gene analysis, six
DNA fragments encoding the PTTH gene (Accession no.
AB186492) promoter from nucleotides )208 to +52, )180
to +52, )167 to +52, ) 119 to +52, )105 to +52, and
)60 to +52 were PCR-amplified from pPT ⁄ EGFP [11],
which contains the PTTH gene promoter from nucleotides
)879 to +52. The forward primers included a Sal I site,
and the reverse primer included a NcoI site. PCR products
were digested with SalI and NcoI and then inserted into
pPT ⁄ EGFP lacking the promoter region of the PTTH
gene. Recombinant plasmids were sequenced, and recom-
binant AcNPVs were prepared according to the manu-
facturer’s instructions. To create three mutants in the
promoter region between nucleotides )180 and )151 of the
PTTH gene (Fig. 2), we performed PCR amplification
using the same reverse primer described above and one of
three forward primers, each of which encoded a SalI site.
The titers of budded virions were determined using the BD
BacPAK Baculovirus Rapid Titer Kit (BD Biosciences,
Palo Alto, CA, USA).
Reporter gene analysis
Four days after injection with recombinant AcNPV, the
brain–SG complex of larvae and pupae was dissected out in
NaCl ⁄ P
i
and mounted onto a hole-slide glass with 1 : 4
Fluoroguard Antifade reagent (Bio-Rad, Hercules, CA,
USA) in NaCl ⁄ P
i
. EGFP fluorescence was detected using an

ECLIPSE E600 microscope (Nikon Co., Tokyo, Japan)
equipped with a DP50CU digital camera (Olympus Co.,
Tokyo, Japan). Digital images of the brain–SG complex were
scanned using view finder lite, version 1.0 (Pixera Co., Los
Gatos, CA, USA) at a sensitivity of 400 and an exposure of
1 ⁄ 15 s. Using NIH image 1.62 ( />nih-image/), the relative fluorescence intensity of the PTPCs
was determined as the intensity of the individual cells relative
to the mean pixel fluorescence for the entire somata (S) of the
brain. Fluorescence images were converted to grayscale and
inverted into black and white images. An area adjacent to the
area of interest (A) and an area from an image lacking a spe-
cimen (N) were scanned as the background signals. When
PTPCs were not visible, the focal plane was adjusted to
faintly signals on small cells in the same field. The relative
fluorescence intensity was calculated as follows: Relative
fluorescence intensity (%) ¼ 100 · ([(S) – (A) – (N)] ⁄ [(A) –
(N)]) for the virus of interest ⁄ ([(S) – (A) – (N)] ⁄ [(A) – (N)])
for the virus carrying nucleotides )879 to +52 of the PTTH
promoter (v[PT ⁄ EGFP]) [11].
In situ hybridization and immunohistochemistry
In situ hybridization was performed as described by Sato
et al. [25] with some modifications. The procedures prior to
proteinase K treatment were adapted from Shiomi et al.
[11]. Brain–SG complex was treated for 5 min with
10 lgÆmL
)1
proteinase K (Roche, Indianapolis, IN, USA)
and then hybridized with digoxigenin (DIG)-labeled sense
and antisense RNA probes, respectively. The DIG-labeled
RNA probes were prepared with a DIG RNA labeling kit

(Roche) using BmMEF2 cDNA as a template. BmMEF2
cDNA encoding from nucleotides +654 to +854 (Acces-
sion no. AB121093) was amplified by PCR and inserted
into the pCR-XL-TOPO vector (Invitrogen) in sense and
antisense directions from the T7 promoter. DIG-labeled
RNA was detected with an alkaline phosphatase-conjugated
anti-DIG IgG using a DIG nucleic acid detection kit
(Roche). For immunohistochemistry, we used an anti-
PTTH monoclonal IgG (3E5mAb) [8] and an anti-corazo-
nin rabbit polyclonal IgG [14]. The immunoreaction
procedures were adapted from Shiomi et al. [11]. EGFP
fluorescence and anti-PTTH immunofluorescent staining
were detected using a Radiance 2000 confocal microscope
Enhancement of PTTH gene expression by MEF2 K. Shiomi et al.
3860 FEBS Journal 272 (2005) 3853–3862 ª 2005 FEBS
(Bio-Rad). Images were adjusted and assembled in Adobe
photoshop cs (Adobe systems Inc., San Jose, CA, USA).
Gel-mobility shift assay
Cell extract was prepared from a mixture of the brain–SG
complex from day 1, 3, and 5 pupae according to Ueda
and Hirose [26] with some modifications. A double-stran-
ded synthetic oligonucleotide corresponding to the PTTH
promoter encoding nucleotides )180 to )151 (Fig. 2) was
end-labeled with T4 polynucleotide kinase and [
32
P]ATP[c
P] and then used as a probe. Incubation and electrophoresis
were performed according to Ueda and Hirose [27]. The
BmMEF2 (MADS) antibody (Qiagen, Valencia, CA, USA)
was generated by immunizing rabbits with a peptide enco-

ding the 15 N-terminal amino acids of BmMEF2.
Cloning of the B. mori MEF2 (BmMEF2) cDNA
Poly(A)
+
RNA was directly purified from brain–SG complex
of day 3 pupae using Dynabeads oligo(dT)
25
(Dynal Biotech
LLC., Brown Deer, WI, USA). RT-PCR was performed
using degenerate primers based on the sequences of the
MADS box and the MEF2 domain [13] common to several
organisms (Accession nos AB01288, U66569, AJ005425,
BC011070, BC040949, AJ002238, U66570, Z19124, X83527,
D49970, and U36198): 5¢-CAGGTGACCTTYAMCA-
ARMG-3¢ (forward) and 5¢-TCRTGDGGYTCRTTR-
TAYTC-3¢ (reverse). The full-length cDNA sequence was
determined using a SMART RACE cDNA amplification kit
(Clontech, Mountain View, CA, USA). Finally, the full-
length BmMEF2 cDNA (Accession no. AB121093) was
amplified by RT-PCR.
RT-PCR and Southern hybridization
Eggs were collected 2 h and 3, 5, and 9 days after oviposi-
tion. Various tissues were dissected from day 4 fifth instar
larvae and day 3 pupae. Total RNAs were extracted from
eggs and various tissues using TRIzol reagent (Invitrogen)
and then subjected to poly(A)
+
RNA purification using
Dynabeads oligo(dT)
25

(Dynal). Poly(A)
+
RNA from the
brain–SG complex was directly purified using Dynabeads
Oligo (dT)
25
(Dynal). First-strand DNA was synthesized
using a SMART RACE amplification kit (Clontech). PCR
amplification was carried out on mRNAs for BmMEF2,
PTTH, and actin A3. The BmMEF2 cDNA was amplified
from nucleotides +654 to +2685 (Accession no.
AB121093), the PTTH cDNA from +34 to +708 (Acces-
sion no. D90082), and the actin A3 cDNA from +70 to
+498 (Accession no. U49854). PCR products were subjec-
ted to electrophoresis, transferred to Hybond-N
+
nylon
membranes (Amersham, Little Chalfont, Bucks, UK), and
then hybridized with the
32
P-labeled internal oligonucleo-
tides encoding nucleotides +908 to +937 of BmMEF2,
+252 to +275 of PTTH, or +308 to +331 of actin A3.
Overexpression and RNA interference for
BmMEF2 mRNA
Two recombinant AcNPVs were constructed for over-
expression (v[PT ⁄ MEFs]) or silencing (v[PT ⁄ MEFi]) of the
BmMEF2 gene. To obtain v[PT ⁄ MEFs], the BmMEF
cDNA corresponding to the open reading frame was inser-
ted downstream of the PTTH promoter. The PCR product

of the BmMEF2 cDNA was ligated to the recombinant
plasmid pPT ⁄ EGFP [11] after excision of the EGFP cDNA
by digestion with NcoI and XhoI. To obtain the v[PT ⁄
MEFi], we constructed two inverted repeat DNAs corres-
ponding to the 1.2-kbp BmMEF2 cDNA fragment from
nucleotides +748 to +1934. These were inserted down-
stream of the PTTH promoter with 200-bp spacer
sequences consisting of the intron sequence (nucleotides
+369 to +568) of the DH-PBAN gene [28] as described by
Giordano et al. [29].
Acknowledgements
This research was funded by grants from the Research
for the Future Program from the Japan Society for the
Promotion of Science (JSPS-RFTF99L01203). Addi-
tional support was provided by Grants-in-Aid
(17688003 and 17658027) from the Ministry of Educa-
tion, Science, Sports and Culture of Japan. We are
also indebted to the Division of Gene Research,
Research Center for Human and Environmental Sci-
ences, Shinshu University, for providing the facilities
for these studies.
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Enhancement of PTTH gene expression by MEF2 K. Shiomi et al.
3862 FEBS Journal 272 (2005) 3853–3862 ª 2005 FEBS