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 t
Trang 1of the expression of the prothoracicotropic hormone gene
in the silkworm, Bombyx mori
Kunihiro Shiomi1, Yoshihiro Fujiwara1, Tsutomu Atsumi1, Zenta Kajiura1, Masao Nakagaki1,
Yoshiaki Tanaka2, Akira Mizoguchi3, Toshinobu Yaginuma4and Okitsugu Yamashita4,5
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: shiomi@giptc.shinshu-u.ac.jp
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 facfac-tor 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.
Trang 2closely 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%, n¼
21 and 95.5 ± 7.7%, n¼ 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%, n¼ 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%, n¼ 37; Fig 1L,L¢) No expression was observed when nucleotides )60 to +52 of the PTTH promoter were used (2.1 ± 4.1%, n¼ 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· 106pfuÆmL)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
Trang 3fifth 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% (n¼ 35) (Fig 1O), 40.1 ± 15.7% (n ¼ 20)
(Fig 1P), and 5.7 ± 5.1% (n¼ 20) (Fig 1Q),
respect-ively Injection with recombinant AcNPVs carrying
nu-cleotides )879, )180, )167, or )105 to +52 of the
PTTHpromoter resulted in hemolymph virus titers of
3.52, 5.01, 2.92 and 4.27· 108pfuÆmL)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 (http://www.genomatix.de/) As shown in Fig 2A, we found that the DNA sequence bound by MEF2, C⁄ TTA(A ⁄ T)4TAG⁄ A [12], is conserved at the 5¢-upstream region from nucleotides )180 to )151 of
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
O L
J
I
K
D
C B
E
M
R
-879 -208 -180 -167 -119 -105 -60
Relative fluorescence intensity (%)
G H I J
K L
M
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.
Trang 4of 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, or M3 sequence
(Fig 2A,C) Based on fluorescence intensity, the M3
virus was as effective (92.7 ± 5.4%, n¼ 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%, n¼
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%, n¼ 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· 106pfuÆmL)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 Oligo-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.
Trang 5+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,
BmMEF2mRNA 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
BmMEF2RNA 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 *.
Trang 6lateral 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
BmMEF2mRNA under control of the PTTH
promo-ter, respectively When injected at 102pfu 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· 106pfuÆmL)1, respectively, and when injected
at 106pfu per pupa, the virus titers were 1.98, 1.18, and 1.57· 107pfuÆmL)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 102pfu 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 106pfu 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 102 and 106pfu 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
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
ActA3
PTTH lane: 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
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/MEFs PT/MEFi
C 102 106 102 106 102 106 (PFU/pupa)
MEF2
PTTH
ActA3
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.
Trang 7We 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
PTTHgene 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)4TAG⁄ 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 Ia1 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 Ia1neurosecretory 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
Trang 8Aizono 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 25C under
con-tinuous darkness Larvae were reared on an artificial diet
(Silkmate-2M, Nosan Co., Yokohama, Japan) at 25–27C
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 25C 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 SalI 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⁄ Pi and mounted onto a hole-slide glass with 1 : 4 Fluoroguard Antifade reagent (Bio-Rad, Hercules, CA, USA) in NaCl⁄ Pi 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 (http://www.rsb.info.nih.gov/ 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
Trang 9(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 [32P]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 32P-labeled internal
oligonucleo-tides encoding nucleooligonucleo-tides +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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